U.S. patent application number 16/584356 was filed with the patent office on 2020-09-03 for tetravalent anti-psgl-1 antibodies and uses thereof.
The applicant listed for this patent is AbGenomics International Inc., BioAlliance C.V.. Invention is credited to Rong-Hwa LIN, Shih-Yao LIN, Yu-Ying TSAI.
Application Number | 20200277395 16/584356 |
Document ID | / |
Family ID | 1000004842807 |
Filed Date | 2020-09-03 |
United States Patent
Application |
20200277395 |
Kind Code |
A1 |
LIN; Rong-Hwa ; et
al. |
September 3, 2020 |
TETRAVALENT ANTI-PSGL-1 ANTIBODIES AND USES THEREOF
Abstract
Provided herein are tetravalent antibodies that specifically
bind to human PSGL-1. Unlike bivalent antibodies, these tetravalent
antibodies contain a dimer of two monomers, with each monomer
comprising two light chain variable (VL) domains and two heavy
chain variable (VH) domains. This format allows for
cross-linker/FcR-expressing cell-independent tetravalent antibodies
against PSGL-1 that show enhanced efficacy as compared to bivalent
PSGL-1 antibodies. These tetravalent antibodies can be used in a
variety of diagnostic and therapeutic methods, including without
limitation treating T-cell mediated inflammatory diseases,
transplantations, and transfusions.
Inventors: |
LIN; Rong-Hwa; (Palo Alto,
CA) ; LIN; Shih-Yao; (Taipei, TW) ; TSAI;
Yu-Ying; (Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioAlliance C.V.
AbGenomics International Inc. |
Amsterdam
Dover |
DE |
NL
US |
|
|
Family ID: |
1000004842807 |
Appl. No.: |
16/584356 |
Filed: |
September 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15400888 |
Jan 6, 2017 |
10472422 |
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16584356 |
|
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62276806 |
Jan 8, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/622 20130101;
C07K 2317/53 20130101; C07K 2317/76 20130101; C07K 2317/626
20130101; C07K 2317/56 20130101; C07K 2317/35 20130101; C07K
2317/21 20130101; C07K 2317/24 20130101; C07K 2317/732 20130101;
C07K 2317/52 20130101; C07K 16/2896 20130101; C07K 2317/565
20130101; A61P 29/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 29/00 20060101 A61P029/00 |
Claims
1. A tetravalent antibody that specifically binds to human PSGL-1,
the tetravalent antibody comprising a dimer of two monomers,
wherein each monomer of the dimer comprises a single-chain
polypeptide comprising, from N-terminus to C-terminus: (a) a first
light chain variable (VL) domain; (b) a first linker sequence; (c)
a first heavy chain variable (VH) domain; (d) a second linker
sequence; (e) a second VL domain; (f) a third linker sequence; (g)
a second VH domain; (h) a fourth linker sequence; and (i) an
antibody Fc domain, wherein each of the first and the second VL
domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; wherein each of
the first and the second VH domains comprises a CDR-H1, a CDR-H2,
and a CDR-H3; and wherein each of the first and the second VL
domains forms a VH-VL binding unit with a corresponding VH domain
of the first and the second VH domains, and wherein each of the two
VH-VL binding units is specific for human PSGL-1.
2. The tetravalent antibody of claim 1, wherein at least one of the
two VH domains comprises: (i) a CDR-H1 comprising the amino acid
sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid
sequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino
acid sequence of SEQ ID NO:19; and/or wherein at least one of the
two VL domains comprises: (i) a CDR-L1 comprising the amino acid
sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid
sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino
acid sequence of SEQ ID NO:22.
3. (canceled)
4. The tetravalent antibody of claim 2, wherein each of the two VH
domains comprises the amino acid sequence of SEQ ID NO:23; an amino
acid sequence having at least 90%, at least 95%, or at least 99%
sequence identity to SEQ ID NO:23; the amino acid sequence of SEQ
ID NO:29; or an amino acid sequence having at least 90%, at least
95%, or at least 99% sequence identity to SEQ ID NO:29; and/or
wherein each of the two VL domains comprises the amino acid
sequence of SEQ ID NO:24; an amino acid sequence having at least
90%, at least 95%, or at least 99% sequence identity to SEQ ID
NO:24; the amino acid sequence of SEQ ID NO:30; or an amino acid
sequence having at least 90%, at least 95%, or at least 99%
sequence identity to SEQ ID NO:30.
5-9. (canceled)
10. The tetravalent antibody of claim 1, wherein the first, second
and third linker sequences each comprise two or more repeats of the
amino acid sequence of SEQ ID NO:25, or the first, second or third
linker sequence comprises the amino acid sequence of SEQ ID NO:33,
34, 35, or 36.
11. (canceled)
12. (canceled)
13. The tetravalent antibody of claim 1, wherein the fourth linker
sequence comprises the amino acid sequence of SEQ ID NO:26.
14. The tetravalent antibody of claim 1, wherein each of the two
single-chain polypeptides comprises the amino acid sequence of SEQ
ID NO:1, or an amino acid sequence having at least 90%, at least
95%, or at least 99% sequence identity to SEQ ID NO:1.
15. (canceled)
16. A tetravalent antibody that specifically binds to human PSGL-1,
the tetravalent antibody comprising a dimer of two monomers,
wherein each monomer of the dimer comprises a single-chain
polypeptide comprising, from N-terminus to C-terminus: (a) a first
heavy chain variable (VH) domain; (b) a first linker sequence; (c)
a first light chain variable (VL) domain; (d) a second linker
sequence; (e) a second VL domain; (f) a third linker sequence; (g)
a second VH domain; (h) a fourth linker sequence; and (i) an
antibody Fc domain, wherein each of the first and the second VL
domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; wherein each of
the first and the second VH domains comprises a CDR-H1, a CDR-H2,
and a CDR-H3; and wherein each of the first and the second VL
domains forms a VH-VL binding unit with a corresponding VH domain
of the first and the second VH domains, and wherein each of the two
VH-VL binding units is specific for human PSGL-1.
17. The tetravalent antibody of claim 16, wherein at least one of
the two VH domains comprises: (i) a CDR-H1 comprising the amino
acid sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino
acid sequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the
amino acid sequence of SEQ ID NO:19; and/or wherein at least one of
the two VL domains comprises: (i) a CDR-L1 comprising the amino
acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino
acid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the
amino acid sequence of SEQ ID NO:22.
18. (canceled)
19. The tetravalent antibody of claim 17, wherein each of the two
VH domains comprises the amino acid sequence of SEQ ID NO:23; an
amino acid sequence having at least 90%, at least 95%, or at least
99% sequence identity to SEQ ID NO:23; the amino acid sequence of
SEQ ID NO:29; or an amino acid sequence having at least 90%, at
least 95%, or at least 99% sequence identity to SEQ ID NO:29;
and/or wherein each of the two VL domains comprises the amino acid
sequence of SEQ ID NO:24; an amino acid sequence having at least
90%, at least 95%, or at least 99% sequence identity to SEQ ID
NO:24; the amino acid sequence of SEQ ID NO:30; or an amino acid
sequence having at least 90%, at least 95%, or at least 99%
sequence identity to SEQ ID NO:30.
20-24. (canceled)
25. The tetravalent antibody of claim 16, wherein the first and the
third linker sequences have the same sequence comprising five
repeats of SEQ ID NO:25.
26. (canceled)
27. The tetravalent antibody of claim 16, wherein the fourth linker
sequence comprises the amino acid sequence of SEQ ID NO:26.
28. The tetravalent antibody of claim 16, wherein each of the two
single-chain polypeptides comprises the amino acid sequence of SEQ
ID NO:3, or an amino acid sequence having at least 90%, at least
95%, or at least 99% sequence identity to SEQ ID NO:3.
29. (canceled)
30. A tetravalent antibody that specifically binds to human PSGL-1,
the tetravalent antibody comprising a dimer of two monomers,
wherein each monomer of the dimer comprises an antibody heavy chain
and an antibody light chain; wherein the antibody light chain
comprises, from N-terminus to C-terminus: (i) a first heavy chain
variable (VH) domain, (ii) a first linker sequence, (iii) a first
light chain variable (VL) domain, (iv) a second linker sequence,
(v) a second VL domain, and (vi) a light chain constant (CL)
domain; wherein the antibody heavy chain comprises: (i) a second VH
domain, and (ii) a heavy chain constant region comprising a first
heavy chain constant region (CH1) domain, an antibody hinge region,
an second heavy chain constant region (CH2) domain, and a third
heavy chain constant region (CH3) domain; wherein each of the first
and the second VL domains comprises a CDR-L1, a CDR-L2, and a
CDR-L3; wherein each of the first and the second VH domains
comprises a CDR-H1, a CDR-H2, and a CDR-H3; and wherein each of the
first and the second VL domains forms a VH-VL binding unit with a
corresponding VH domain of the first and the second VH domains, and
wherein each of the two VH-VL binding units is specific for human
PSGL-1.
31. The tetravalent antibody of claim 30, wherein at least one of
the first and the second VH domains comprises: (i) a CDR-H1
comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2
comprising the amino acid sequence of SEQ ID NO:18; and (iii) a
CDR-H3 comprising the amino acid sequence of SEQ ID NO:19; and/or
wherein at least one of the first and the second VL domains
comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ
ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ
ID NO:21; and (iii) a CDR-L3 comprising the amino acid sequence of
SEQ ID NO:22.
32. (canceled)
33. The tetravalent antibody of claim 31, wherein the first and the
second VH domains each comprise the amino acid sequence of SEQ ID
NO:23; an amino acid sequence having at least 90%, at least 95%, or
at least 99% sequence identity to SEQ ID NO:23; the amino acid
sequence of SEQ ID NO:29; or an amino acid sequence having at least
90%, at least 95%, or at least 99% sequence identity to SEQ ID
NO:29; and/or wherein each of the two VL domains comprises the
amino acid sequence of SEQ ID NO:24; an amino acid sequence having
at least 90%, at least 95%, or at least 99% sequence identity to
SEQ ID NO:24; the amino acid sequence of SEQ ID NO:30; or an amino
acid sequence having at least 90%, at least 95%, or at least 99%
sequence identity to SEQ ID NO:30.
34-38. (canceled)
39. The tetravalent antibody of claim 30, wherein the CL domain is
a kappa CL domain.
40. The tetravalent antibody of claim 30, wherein the first linker
sequence comprises five repeats of SEQ ID NO:25; and/or wherein the
second linker sequence comprises the amino acid sequence of SEQ ID
NO:28.
41. (canceled)
42. The tetravalent antibody of claim 30, wherein the antibody
light chain comprises the amino acid sequence of SEQ ID NO:7, or an
amino acid sequence having at least 90%, at least 95%, or at least
99% sequence identity to SEQ ID NO:7; and/or wherein the antibody
heavy chain comprises the amino acid sequence of SEQ ID NO:11, or
an amino acid sequence having at least 90%, at least 95%, or at
least 99% sequence identity to SEQ ID NO:11.
43-45. (canceled)
46. The tetravalent antibody of claim 1, wherein the antibody Fc
domain is a human antibody Fc domain.
47-49. (canceled)
50. An isolated polynucleotide encoding the tetravalent antibody of
claim 1.
51. (canceled)
52. A vector comprising the isolated polynucleotide of claim
50.
53. A host cell comprising the polynucleotide of claim 50.
54. A method of producing a tetravalent antibody comprising
culturing the host cell of claim 53 so that the tetravalent
antibody is produced.
55. (canceled)
56. A pharmaceutical composition comprising the tetravalent
antibody of claim 1 and a pharmaceutically acceptable carrier.
57-63. (canceled)
64. A method of treating a T-cell mediated inflammatory disease,
the method comprising administering to a subject in need thereof a
therapeutically effective amount of the tetravalent antibody of
claim 1.
65. A method for treating an individual in need of a transfusion or
transplantation, comprising administering to the individual a
therapeutically effective amount of the tetravalent antibody of
claim 1 before, concurrently with, and/or after the transfusion or
transplantation.
66-70. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. No. 62/276,806, filed Jan. 8, 2016,
which is incorporated herein by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
606592001300SEQLIST.TXT, date recorded: Jan. 3, 2017, size: 70
KB).
FIELD
[0003] Provided herein are tetravalent antibodies that specifically
bind to human P-selectin glycoprotein ligand-1 (PSGL-1), as well as
polynucleotides, vectors, host cells, methods, pharmaceutical
compositions, kits, and uses related thereto. These tetravalent
antibodies may find use in a variety of diagnostic and therapeutic
methods, including without limitation treating T-cell mediated
inflammatory diseases, transplantations, and transfusions.
BACKGROUND
[0004] Inflammatory responses to infection or injury are initiated
by the adherence of leukocytes to the vascular wall (McEver et al,
1997, J. Clin. Invest., 100 (3): 485-492). Selectin represents a
family of glycoproteins which mediate the first
leukocyte-endothelial cell and leukocyte-platelet interactions
during inflammation. The selectin family, which consists of
L-selectin, E-selectin, and P-selectin, comprises an NH2-terminal
lectin domain, followed by an EGF-like domain, a series of
consensus repeats, a transmembrane domain, and a short cytoplasmic
tail. The lectin domains of selectins interact with specific
glycoconjugate ligands in order to facilitate cell adhesion.
L-selectin, expressed on most leukocytes, binds to ligands on some
endothelial cells and other leukocytes. E-selectin, expressed on
cytokine activated endothelial cells, binds to ligands on most
leukocytes. P-selectin, expressed on activated platelets and
endothelial cells, also binds to ligands on most leukocytes.
[0005] P-selectin glycoprotein ligand-1 ("PSGL-1"), also known as
SELPLG or CD162 (cluster of differentiation 162) is a human
mucin-type glycoprotein ligand for all three selectins (Constantin,
Gabriela, 2004, Drug News Perspect., 17(9): 579-585; McEver et al.,
1997, J. Clin. Invest., 100 (3): 485-492). PSGL-1 is a
disulfide-bonded homodimer with two 120-kD subunits and is
expressed on the surface of monocytes, lymphocytes, granulocytes,
and in some CD34.sup.+ stem cells. PSGL-1 is likely to contribute
to pathological leukocyte recruitment in many inflammatory
disorders since it facilitates the adhesive interactions of
selectins. In addition, PSGL-1 is shown to have a unique regulatory
role in T cells. Mice deficient in PSGL-1 show enhanced
proliferative responses and autoimmunity, suggesting that PSGL-1
plays an important role in down-regulating T cell responses
(Krystle M. et al. J. Immunol. 2012; 188:1638-1646. Urzainqui et
al. Ann Rheum Dis 2013; 71:650; Perez-Frias A, et al. Arthritis
Rheumatol. 2014 November; 66(11):3178-89.; Angiari et al. J
Immunol. 2013; 191(11):5489-500).
[0006] Several anti-PSGL-1 antibodies have been developed (see,
e.g., International Application Pub. Nos. WO 2005/110475, WO
2003/013603, and WO 2012/174001; Constantin, Gabriela, 2004, Drug
News Perspect., 17(9): 579-585, Chen et al. Blood. 2004;
104(10):3233-42, Huang et al, Eur J Immunol. 2005; 35(7):2239-49;
and U.S. Pat. No. 7,604,800). Some of the existing agonistic PSGL-1
antibodies preferentially induce apoptosis of late-stage activated
T cells but not other PSGL-1-expressing cells; such antibodies may
therefore be useful as anti-inflammatory therapeutics, or for use
in transplantations and/or transfusions. However, a need exists for
improved anti-PSGL-1 antibodies with greater in vivo efficacy than
existing antibodies.
[0007] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
BRIEF SUMMARY
[0008] To meet this need, provided herein are tetravalent
antibodies that specifically bind to human PSGL-1, as well as
polynucleotides, vectors, host cells, methods, pharmaceutical
compositions, kits, and uses related thereto. The present
disclosure demonstrates that tetravalent antibodies that
specifically bind to human PSGL-1 have greater potency and efficacy
than conventional (e.g., bivalent) anti-PSGL-1 antibodies. As such,
these tetravalent antibodies may find use, inter alia, in
diagnostic and/or therapeutic methods, uses, and compositions
related to T-cell function, such as in treating T-cell mediated
inflammatory diseases, transfusions, and/or transplantations.
[0009] Accordingly, in one aspect, provided herein is a tetravalent
antibody that specifically binds to human PSGL-1, the tetravalent
antibody comprising a dimer of two monomers, wherein each monomer
of the dimer comprises a single-chain polypeptide comprising: (a)
two light chain variable (VL) domains, wherein each of the two VL
domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; (b) two heavy
chain variable (VH) domains, wherein each of the two VH domains
comprises a CDR-H1, a CDR-H2, and a CDR-H3; and (c) an antibody Fc
domain, wherein each of the two VL domains forms a VH-VL binding
unit with a corresponding VH domain of the two VH domains, and
wherein each of the two VH-VL binding units is specific for human
PSGL-1. In some embodiments, at least one of the two VH domains
comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ
ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ
ID NO:18; and (iii) a CDR-H3 comprising the amino acid sequence of
SEQ ID NO:19. In some embodiments, each of the two VH domains
comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ
ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ
ID NO:18; and (iii) a CDR-H3 comprising the amino acid sequence of
SEQ ID NO:19. In some embodiments, one or both of the two VH
domains comprises the amino acid sequence of SEQ ID NO:23, or an
amino acid sequence having at least 90%, at least 95%, or at least
99% sequence identity to SEQ ID NO:23. In some embodiments, one or
both of the two VH domains comprises the amino acid sequence of SEQ
ID NO:29, or an amino acid sequence having at least 90%, at least
95%, or at least 99% sequence identity to SEQ ID NO:29. In some
embodiments, at least one of the two VL domains comprises: (i) a
CDR-L1 comprising the amino acid sequence of SEQ ID NO:20; (ii) a
CDR-L2 comprising the amino acid sequence of SEQ ID NO:21; and
(iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:22.
In some embodiments, each of the two VL domains comprises: (i) a
CDR-L1 comprising the amino acid sequence of SEQ ID NO:20; (ii) a
CDR-L2 comprising the amino acid sequence of SEQ ID NO:21; and
(iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:22.
In some embodiments, one or both of the two VL domains comprises
the amino acid sequence of SEQ ID NO:24, or an amino acid sequence
having at least 90%, at least 95%, or at least 99% sequence
identity to SEQ ID NO:24. In some embodiments, one or both of the
two VL domains comprises the amino acid sequence of SEQ ID NO:30,
or an amino acid sequence having at least 90%, at least 95%, or at
least 99% sequence identity to SEQ ID NO:30. In some embodiments,
each of the two single-chain polypeptides comprises, from
N-terminus to C-terminus: (a) a first VL domain of the two VL
domains; (b) a first linker sequence; (c) a first VH domain of the
two VH domains; (d) a second linker sequence; (e) a second VL
domain of the two VL domains; (f) a third linker sequence; (g) a
second VH domain of the two VH domains; (h) a fourth linker
sequence; and (i) the antibody Fc domain. In some embodiments, the
first, second and third linker sequences each comprise two or more
repeats of the amino acid sequence of SEQ ID NO:25. In some
embodiments, the first and the third linker sequences have the same
sequence and comprise two repeats of SEQ ID NO:25. In some
embodiments, the second linker sequence comprises five repeats of
SEQ ID NO:25. In some embodiments, the fourth linker sequence
comprises the amino acid sequence of SEQ ID NO:26. In some
embodiments, each of the two single-chain polypeptides comprises
the amino acid sequence of SEQ ID NO:1, or an amino acid sequence
having at least 90%, at least 95%, or at least 99% sequence
identity to SEQ ID NO:1. In some embodiments, each of the two
single-chain polypeptides is encoded by a polynucleotide comprising
the polynucleotide sequence of SEQ ID NO:2. In some embodiments,
each of the two single-chain polypeptides comprises, from
N-terminus to C-terminus: (a) a first VH domain of the two VH
domains; (b) a first linker sequence; (c) a first VL domain of the
two VL domains; (d) a second linker sequence; (e) a second VL
domain of the two VL domains; (f) a third linker sequence; (g) a
second VH domain of the two VH domains; (h) a fourth linker
sequence; and (i) the antibody Fc domain. In some embodiments, each
of the two single-chain polypeptides comprises, from N-terminus to
C-terminus: (a) a first VL domain of the two VL domains; (b) a
first linker sequence; (c) a first VH domain of the two VH domains;
(d) a second linker sequence; (e) a second VH domain of the two VH
domains; (f) a third linker sequence; (g) a second VL domain of the
two VL domains; (h) a fourth linker sequence; and (i) the antibody
Fc domain. In some embodiments, the first, second or third linker
sequence comprises two or more repeats of the amino acid sequence
of SEQ IN NO:25. In some embodiments, the first, second or third
linker sequence comprises the amino acid sequence of SEQ ID NO:33,
34, 35, or 36. In some embodiments, the first and the third linker
sequences have the same sequence comprising five repeats of SEQ ID
NO:25. In some embodiments, the second linker sequence comprises
the amino acid sequence of SEQ ID NO:27. In some embodiments, the
fourth linker sequence comprises the amino acid sequence of SEQ ID
NO:26. In some embodiments, each of the two single-chain
polypeptides comprises the amino acid sequence of SEQ ID NO:3, or
an amino acid sequence having at least 90%, at least 95%, or at
least 99% sequence identity to SEQ ID NO:3. In some embodiments,
each of the two single-chain polypeptides is encoded by a
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:4. In some embodiments, each of the two single-chain
polypeptides comprises the amino acid sequence of SEQ ID NO:5. In
some embodiments, each of the two single-chain polypeptides is
encoded by a polynucleotide comprising the polynucleotide sequence
of SEQ ID NO:6.
[0010] In another aspect, provided herein is a tetravalent antibody
that specifically binds to human PSGL-1, the tetravalent antibody
comprising a dimer of two monomers, wherein each monomer of the
dimer comprises an antibody heavy chain and an antibody light
chain; wherein the antibody light chain comprises: (i) two light
chain variable (VL) domains, wherein each of the two VL domains
comprises a CDR-L1, a CDR-L2, and a CDR-L3, (ii) a first heavy
chain variable (VH) domain, and (iii) a light chain constant (CL)
domain; wherein the antibody heavy chain comprises: (i) a second
heavy chain variable (VH) domain, and (ii) a heavy chain constant
region comprising a first heavy chain constant region (CH1) domain,
an antibody hinge region, an second heavy chain constant region
(CH2) domain, and a third heavy chain constant region (CH3) domain;
wherein the first and the second VH domains each comprise a CDR-H1,
a CDR-H2, and a CDR-H3, wherein each of the two VL domains forms a
VH-VL binding unit with a corresponding VH domain of the first and
the second VH domains, and wherein each of the two VH-VL binding
units is specific for human PSGL-1. In some embodiments, at least
one of the first and the second VH domains comprises: (i) a CDR-H1
comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2
comprising the amino acid sequence of SEQ ID NO:18; and (iii) a
CDR-H3 comprising the amino acid sequence of SEQ ID NO:19. In some
embodiments, the first and the second VH domains each comprise: (i)
a CDR-H1 comprising the amino acid sequence of SEQ ID NO:17; (ii) a
CDR-H2 comprising the amino acid sequence of SEQ ID NO:18; and
(iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:19.
In some embodiments, the first and/or the second VH domains
comprise the amino acid sequence of SEQ ID NO:23, or an amino acid
sequence having at least 90%, at least 95%, or at least 99%
sequence identity to SEQ ID NO:23. In some embodiments, the first
and/or the second VH domains comprise the amino acid sequence of
SEQ ID NO:29, or an amino acid sequence having at least 90%, at
least 95%, or at least 99% sequence identity to SEQ ID NO:29. In
some embodiments, at least one of the first and the second VL
domains comprises: (i) a CDR-L1 comprising the amino acid sequence
of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence
of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino acid
sequence of SEQ ID NO:22. In some embodiments, the first and the
second VL domains each comprise: (i) a CDR-L1 comprising the amino
acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino
acid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the
amino acid sequence of SEQ ID NO:22. In some embodiments, the first
and/or the second VL domains comprise the amino acid sequence of
SEQ ID NO:24, or an amino acid sequence having at least 90%, at
least 95%, or at least 99% sequence identity to SEQ ID NO:24. In
some embodiments, the first and/or the second VL domains comprise
the amino acid sequence of SEQ ID NO:30, or an amino acid sequence
having at least 90%, at least 95%, or at least 99% sequence
identity to SEQ ID NO:30. In some embodiments, the antibody light
chain comprises, from N-terminus to C-terminus: (a) the first VH
domain; (b) a first linker sequence; (c) a first VL domain of the
two or more VL domains; (d) a second linker sequence; (e) a second
VL domain of the two or more VL domains; and (f) the CL domain. In
some embodiments, the CL domain is a kappa CL domain. In some
embodiments, the first linker sequence comprises five repeats of
SEQ ID NO:25. In some embodiments, the second linker sequence
comprises the amino acid sequence of SEQ ID NO:28. In some
embodiments, the antibody light chain comprises the amino acid
sequence of SEQ ID NO:7, or an amino acid sequence having at least
90%, at least 95%, or at least 99% sequence identity to SEQ ID
NO:7. In some embodiments, the antibody light chain is encoded by a
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:8. In some embodiments, the antibody light chain comprises, from
N-terminus to C-terminus: (a) a first VL domain of the two VL
domains; (b) the CL domain; (c) a first linker sequence; (d) the
first VH domain; (e) a second linker sequence; and (f) a second VL
domain of the two VL domains. In some embodiments, the CL domain is
a kappa CL domain. In some embodiments, the first linker sequence
comprises two repeats of SEQ ID NO:25. In some embodiments, the
second linker sequence comprises five repeats of SEQ ID NO:25. In
some embodiments, the antibody light chain comprises the amino acid
sequence of SEQ ID NO:9. In some embodiments, the antibody light
chain is encoded by a polynucleotide comprising the polynucleotide
sequence of SEQ ID NO:10. In some embodiments, the antibody heavy
chain comprises, from N-terminus to C-terminus: (a) the second VH
domain; and (b) a heavy chain constant region comprising a first
heavy chain constant region (CH1) domain, an antibody hinge region,
an second heavy chain constant region (CH2) domain, and a third
heavy chain constant region (CH3) domain. In some embodiments, the
antibody heavy chain comprises the amino acid sequence of SEQ ID
NO:11, or an amino acid sequence having at least 90%, at least 95%,
or at least 99% sequence identity to SEQ ID NO:11. In some
embodiments, the antibody heavy chain is encoded by a
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:12.
[0011] In another aspect, provided herein is a tetravalent antibody
that specifically binds to human PSGL-1, the tetravalent antibody
comprising a dimer of two monomers, wherein each monomer of the
dimer comprises an antibody heavy chain and an antibody light
chain; wherein the antibody light chain comprises: (i) a first
heavy chain variable (VH) domain, (ii) a first light chain variable
(VL) domain, and (iii) a light chain constant (CL) domain; wherein
the antibody heavy chain comprises: (i) a second heavy chain
variable (VH) domain, (ii) a second light chain variable (VL)
domain, and (iii) a heavy chain constant domain comprising a first
heavy chain constant region (CH1) domain, an antibody hinge region,
an second heavy chain constant region (CH2) domain, and a third
heavy chain constant region (CH3) domain; wherein each of the first
and second VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3;
wherein each of the first and second VH domains comprises a CDR-H1,
a CDR-H2, and a CDR-H3; wherein each of the first and second VL
domains forms a VH-VL binding unit with a corresponding VH domain
of the first and second VH domains; and wherein each of the two
VH-VL binding units is specific for human PSGL-1. In some
embodiments, at least one of the first and second VH domains
comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ
ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ
ID NO:18; and (iii) a CDR-H3 comprising the amino acid sequence of
SEQ ID NO:19. In some embodiments, the first and the second VH
domains each comprise: (i) a CDR-H1 comprising the amino acid
sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid
sequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino
acid sequence of SEQ ID NO:19. In some embodiments, the first
and/or the second VH domains comprise the amino acid sequence of
SEQ ID NO:23. In some embodiments, the first and/or the second VH
domains comprise the amino acid sequence of SEQ ID NO:29. In some
embodiments, at least one of the first and second VL domains
comprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ
ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ
ID NO:21; and (iii) a CDR-L3 comprising the amino acid sequence of
SEQ ID NO:22. In some embodiments, the first and the second VL
domains each comprise: (i) a CDR-L1 comprising the amino acid
sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid
sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino
acid sequence of SEQ ID NO:22. In some embodiments, the first
and/or the second VL domains comprise the amino acid sequence of
SEQ ID NO:24. In some embodiments, the first and/or the second VL
domains comprise the amino acid sequence of SEQ ID NO:30. In some
embodiments, the antibody light chain comprises, from N-terminus to
C-terminus: (a) the first VH domain; (b) a first linker sequence;
(c) the first VL domain; and (d) the CL domain. In some
embodiments, the CL domain is a kappa CL domain. In some
embodiments, the first linker sequence comprises five repeats of
SEQ ID NO:25. In some embodiments, the antibody light chain
comprises the amino acid sequence of SEQ ID NO:13. In some
embodiments, the antibody light chain is encoded by a
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:14. In some embodiments, the antibody heavy chain comprises,
from N-terminus to C-terminus: (a) the second VH domain; (b) a
second linker sequence; (c) the second VL domain; and (d) the heavy
chain constant region comprising the first heavy chain constant
region (CH1) domain, the antibody hinge region, the second heavy
chain constant region (CH2) domain, and the third heavy chain
constant region (CH3) domain. In some embodiments, the second
linker sequence comprises five repeats of SEQ ID NO:25. In some
embodiments, the antibody heavy chain comprises the amino acid
sequence of SEQ ID NO:15. In some embodiments, the antibody heavy
chain is encoded by a polynucleotide comprising the polynucleotide
sequence of SEQ ID NO:16.
[0012] In some embodiments of any of the above embodiments, the
antibody Fc domain is a human antibody Fc domain. In some
embodiments, the antibody Fc domain is a human IgG4 Fc domain. In
some embodiments, the human IgG4 Fc domain comprises a hinge region
sequence comprising one or more amino acid substitutions that
result in reduced IgG4 shuffling, as compared to an IgG4 hinge
region lacking the one or more amino acid substitutions. In some
embodiments, the human IgG4 Fc domain comprises a hinge region
sequence comprising a serine to proline substitution at amino acid
228, numbering according to EU index. In some embodiments of any of
the above embodiments, the antibody hinge region comprises a serine
to proline substitution at amino acid 228, numbering according to
EU index. In some embodiments, a tetravalent antibody of the
present disclosure displays enhanced induction of apoptosis in a
target cell (e.g., a cell expressing human PSGL-1 or an epitope
thereof) as compared to a conventional (e.g., bivalent) antibody
having one or more VH or VL domains in common with the tetravalent
antibody. In some embodiments, a tetravalent antibody of the
present disclosure displays enhanced inhibition of DTH (e.g., in a
trans vivo animal model) as compared to a conventional (e.g.,
bivalent) antibody having one or more VH or VL domains in common
with the tetravalent antibody.
[0013] In another aspect, provided herein is an isolated
polynucleotide encoding the tetravalent antibody of any one of the
above embodiments. In some embodiments, the isolated polynucleotide
comprises a polynucleotide sequence selected from the group
consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. In another
aspect, provided herein is a vector comprising the isolated
polynucleotide of any of the above embodiments. In another aspect,
provided herein is a host cell comprising the polynucleotide of any
of the above embodiments and/or the vector of any of the above
embodiments. In another aspect, provided herein is a method of
producing a tetravalent antibody comprising culturing the host cell
of any of the above embodiments so that the tetravalent antibody is
produced. In some embodiments, the method further comprises
recovering the tetravalent antibody from the host cell.
[0014] In another aspect, provided herein is a pharmaceutical
composition comprising the tetravalent antibody of any one of the
above embodiments and a pharmaceutically acceptable carrier. In
another aspect, provided herein is a kit comprising the tetravalent
antibody of any one of the above embodiments and an optional
pharmaceutically acceptable carrier. In some embodiments, the kit
further comprises a package insert comprising instructions for
administration of the tetravalent antibody to treat a T-cell
mediated inflammatory disease or condition. In some embodiments,
the kit further comprises a package insert comprising instructions
for administration of the tetravalent antibody before, concurrently
with, and/or after a transfusion or transplantation. In another
aspect, provided herein is the tetravalent antibody of any one of
the above embodiments for use in treating a T-cell mediated
inflammatory disease or condition. In another aspect, provided
herein is the tetravalent antibody of any one of the above
embodiments for use in treating an individual in need of a
transfusion or transplantation. In another aspect, provided herein
is a use of the tetravalent antibody of any one of the above
embodiments in the manufacture of a medicament for treating a
T-cell mediated inflammatory disease or condition. In another
aspect, provided herein is a use of the tetravalent antibody of any
one of the above embodiments in the manufacture of a medicament for
treating an individual in need of a transfusion or transplantation.
In another aspect, provided herein is a method of treating a T-cell
mediated inflammatory disease or condition, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of the tetravalent antibody of any one of the
above embodiments. In another aspect, provided herein is a method
for treating an individual in need of a transfusion or
transplantation, comprising administering to the individual a
therapeutically effective amount of the tetravalent antibody of any
one of the above embodiments before, concurrently with, and/or
after the transfusion or transplantation. In some embodiments, the
T-cell mediated inflammatory disease is an autoimmune disease. In
some embodiments, the T-cell mediated inflammatory disease is
selected from the group consisting of psoriasis, psoriatic
arthritis, rheumatoid arthritis, Crohn's disease, ankylosing
spondylitis, type I diabetes, ulcerative colitis, multiple
sclerosis, and graft versus host disease (GVHD). In some
embodiments, the psoriasis is plaque psoriasis, chronic plaque
psoriasis, guttate psoriasis, inverse psoriasis, pustular
psoriasis, or erythrodermic psoriasis. In some embodiments, the
transplantation is a transplantation of a tissue selected from the
group consisting of bone marrow, kidney, heart, liver, neuronal
tissue, lung, pancreas, skin, and intestine. In some embodiments,
the transfusion is a transfusion comprising one or more of white
blood cells, red blood cells, and platelets.
[0015] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A & 1B provide schematics illustrating exemplary
tetravalent antibodies in accordance with some embodiments. FIG. 1A
illustrates the following exemplary formats: (1) a dimer composed
of two single-chain diabodies fused to an Fc domain
(scDb.sub.2-Fc), showing linker sequences: (GGGGS).sub.5 (SEQ ID
NO:33), GGGGSAAA (SEQ ID NO:26) and (GGGGS).sub.2 (SEQ ID
NO:34)/(GGGGS).sub.2G (SEQ ID NO:35)/(GGGGS).sub.2GG (SEQ ID
NO:36); (2) two different formats, each having a dimer of two
tandem single-chain variable fragment units (taFv.sub.2-Fc),
showing identical linker sequences for both formats: (GGGGS).sub.5
(SEQ ID NO:33), ASTGS (SEQ ID NO:27), GGGGSAAA (SEQ ID NO:26); and
(3) three different formats based on single-chain variable
fragments (scFv-IgG), showing: scFv.sub.2-LC-IgG4p linker sequences
(GGGGS).sub.5 (SEQ ID NO:33) and ASTGSG.sub.4S (SEQ ID NO:28),
LC-scFv.sub.2-IgG4p linker sequences (GGGGS).sub.2 (SEQ ID NO:34)
and (GGGGS).sub.5 (SEQ ID NO:33), scFv.sub.4-crlG4p linker
sequences (GGGGS).sub.5 (SEQ ID NO:33). FIG. 1B provides another
illustration of the three scFv-based formats, with the variable
fragments shaded and V2 scFvs indicated.
[0017] FIGS. 2A-2C show the verification of the molecular weights
and basic structures of exemplary tetravalent antibodies by
SDS-PAGE followed by Coomassie blue staining. Non-reducing (FIGS.
2A & 2B) and reducing (FIG. 2C) conditions are shown.
DETAILED DESCRIPTION
[0018] Provided herein are tetravalent antibodies that specifically
bind to human PSGL-1. The present disclosure is based at least in
part on the finding described herein that certain tetravalent
anti-PSGL-1 antibodies show enhanced efficacy compared to the
parental anti-PSGL-1 antibody both in vitro and trans vivo. These
tetravalent antibodies displayed higher potency for apoptosis
induction and enhanced efficacy in a trans vivo model for delayed
type hypersensitivity (DTH) than the parental anti-PSGL-1 antibody.
Further provided herein are isolated polynucleotides, vectors, host
cells, pharmaceutical compositions, kits, uses, and methods related
to the tetravalent antibodies. For example, the tetravalent
antibodies of the present disclosure may find use in treating a
T-cell mediated inflammatory disease, or administration before,
concurrently with, and/or after a transfusion or
transplantation.
[0019] In some embodiments, the tetravalent antibodies of the
present disclosure comprise a dimer of two monomers, wherein each
monomer of the dimer comprises a single-chain polypeptide
comprising: (a) two light chain variable (VL) domains, wherein each
of the two VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3;
(b) two heavy chain variable (VH) domains, wherein each of the two
VH domains comprises a CDR-H1, a CDR-H2, and a CDR-H3; and (c) an
antibody Fc domain, wherein each of the two VL domains forms a
VH-VL binding unit with a corresponding VH domain of the two VH
domains, and wherein each of the two VH-VL binding units is
specific for human PSGL-1. In other embodiments, the tetravalent
antibodies of the present disclosure comprise a dimer of two
monomers, wherein each monomer of the dimer comprises an antibody
heavy chain and an antibody light chain; wherein the antibody light
chain comprises: (i) two light chain variable (VL) domains, wherein
each of the two VL domains comprises a CDR-L1, a CDR-L2, and a
CDR-L3, (ii) a first heavy chain variable (VH) domain, and (iii) a
light chain constant (CL) domain; wherein the antibody heavy chain
comprises: (i) a second heavy chain variable (VH) domain, and (ii)
a heavy chain constant region comprising a first heavy chain
constant region (CH1) domain, an antibody hinge region, an second
heavy chain constant region (CH2) domain, and a third heavy chain
constant region (CH3) domain; wherein the first and the second VH
domains each comprise a CDR-H1, a CDR-H2, and a CDR-H3, wherein
each of the two VL domains forms a VH-VL binding unit with a
corresponding VH domain of the first and the second VH domains, and
wherein each of the two VH-VL binding units is specific for human
PSGL-1. In other embodiments, the tetravalent antibodies of the
present disclosure comprise a dimer of two monomers, wherein each
monomer of the dimer comprises an antibody heavy chain and an
antibody light chain; wherein the antibody light chain comprises:
(i) a first heavy chain variable (VH) domain, (ii) a first light
chain variable (VL) domain, and (iii) a light chain constant (CL)
domain; wherein the antibody heavy chain comprises: (i) a second
heavy chain variable (VH) domain, (ii) a second light chain
variable (VL) domain, and (iii) a heavy chain constant region
comprising a first heavy chain constant region (CH1) domain, an
antibody hinge region, an second heavy chain constant region (CH2)
domain, and a third heavy chain constant region (CH3) domain;
wherein each of the first and second VL domains comprises a CDR-L1,
a CDR-L2, and a CDR-L3; wherein each of the first and second VH
domains comprises a CDR-H1, a CDR-H2, and a CDR-H3; wherein each of
the first and second VL domains forms a VH-VL binding unit with a
corresponding VH domain of the first and second VH domains; and
wherein each of the two VH-VL binding units is specific for human
PSGL-1.
I. Definitions
[0020] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also
polypeptides comprising fragments thereof (such as Fab, Fab',
F(ab').sub.2, Fv); single-chain variable fragments (scFv),
single-chain diabodies (scDbs), tandem single-chain variable
fragment (scFv) units (termed taFv for tandem scFv), and mutants or
other configurations thereof; fusion proteins comprising an
antibody portion; and any other modified configuration of the
immunoglobulin molecule that comprises an antigen recognition
site.
[0021] As used herein, a "tetravalent" antibody may refer to an
antibody that comprises four antibody VH-VL binding units, with
each VH-VL binding unit comprising an antibody VH domain and an
antibody VL domain. As used herein, references to a "monomer" of a
tetravalent antibody may include both single-chain polypeptides and
multiple-chain polypeptides. For example, a monomer may refer to a
single-chain polypeptide, or it may refer to an antibody heavy
chain-light chain unit, where the heavy chain and light chain are
encoded by separate polynucleotides and/or are formed from the
association of separate polypeptides.
[0022] An antibody includes an antibody of any class, such as IgG,
IgA, or IgM (or sub-class thereof), and the antibody need not be of
any particular class. Depending on the antibody amino acid sequence
of the constant domain of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains
that correspond to the different classes of immunoglobulins are
called alpha, delta, epsilon, gamma, and mu, respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0023] The antibodies of the present disclosure are further
intended to include bispecific, multispecific, chimeric, humanized,
and recombinantly constructed molecules having affinity for a
polypeptide conferred by at least one CDR region of the antibody.
Single domain antibodies which are either the variable domain of an
antibody heavy chain or the variable domain of an antibody light
chain are known in the art. See, e.g., Holt et al., Trends
Biotechnol. 21:484-490, 2003. Methods of making antibodies
comprising either the variable domain of an antibody heavy chain or
the variable domain of an antibody light chain, containing three of
the six naturally occurring complementarity determining regions
from an antibody, are also known in the art. See, e.g.,
Muyldermans, Rev. Mol. Biotechnol. 74:277-302, 2001.
[0024] As used herein, "monoclonal antibody" refers to an antibody
of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are generally highly specific, being
directed against a single antigenic site. Furthermore, in contrast
to polyclonal antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present disclosure may be made by the hybridoma method first
described by Kohler and Milstein, 1975, Nature, 256:495, or may be
made by recombinant DNA methods such as described in U.S. Pat. No.
4,816,567. The monoclonal antibodies may also be isolated from
phage libraries generated using the techniques described in
McCafferty et al., 1990, Nature, 348:552-554, for example.
[0025] As used herein, a "chimeric antibody" refers to an antibody
having a variable region or part of a variable region from a first
species and a constant region from a second species. An intact
chimeric antibody comprises two copies of a chimeric light chain
and two copies of a chimeric heavy chain. The production of
chimeric antibodies is known in the art (Cabilly et al. (1984),
Proc. Natl. Acad. Sci. USA, 81:3273-3277; Harlow and Lane (1988),
Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory).
Typically, in these chimeric antibodies, the variable region of
both light and heavy chains mimics the variable regions of
antibodies derived from one species of mammal, while the constant
portions are homologous to the sequences in antibodies derived from
another. One clear advantage to such chimeric forms is that, for
example, the variable regions can conveniently be derived from
presently known sources using readily available hybridomas or
B-cells from non-human host organisms in combination with constant
regions derived from, for example, human cell preparations. While
the variable region has the advantage of ease of preparation, and
the specificity is not affected by its source, the constant region
being human is less likely to elicit an immune response from a
human subject when the antibodies are injected than would the
constant region from a non-human source. However, the definition is
not limited to this particular example. In some embodiments, amino
acid modifications are made in the variable and/or constant
region.
[0026] As used herein, "humanized" antibodies refer to forms of
non-human (e.g., murine) antibodies that are specific chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2, or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat, or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (e.g., an Fc domain), typically that of a human
immunoglobulin. Antibodies may have Fc regions modified as
described in WO 99/58572. Other forms of humanized antibodies have
one or more CDRs (one, two, three, four, five, or six) which are
altered with respect to the original antibody, which are also
termed one or more CDRs "derived from" one or more CDRs from the
original antibody.
[0027] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody produced
by a human and/or has been made using any of the techniques for
making human antibodies known in the art or disclosed herein. This
definition of a human antibody includes antibodies comprising at
least one human heavy chain polypeptide or at least one human light
chain polypeptide. One such example is an antibody comprising
murine light chain and human heavy chain polypeptides. Human
antibodies can be produced using various techniques known in the
art. In one embodiment, the human antibody is selected from a phage
library, where that phage library expresses human antibodies
(Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et
al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991,
J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,
222:581). Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016. Alternatively, the human antibody may be prepared by
immortalizing human B-lymphocytes that produce an antibody directed
against a target antigen (such B-lymphocytes may be recovered from
an individual or may have been immunized in vitro). See, e.g., Cole
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77 (1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and
U.S. Pat. No. 5,750,373.
[0028] A "variable region" (the term "variable domain" may be used
interchangeably herein) of an antibody refers to the variable
region of the antibody light chain (VL) or the variable region of
the antibody heavy chain (VH), either alone or in combination. The
variable regions of the heavy and light chain (VH and VL domains,
respectively) each consist of four framework regions (FR) connected
by three complementarity determining regions (CDRs) also known as
hypervariable regions. The CDRs in each chain are held together in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen-binding site of
antibodies. There are at least two techniques for determining CDRs:
(1) an approach based on cross-species sequence variability (i.e.,
Kabat et al. Sequences of Proteins of Immunological Interest, (5th
ed., 1991, National Institutes of Health, Bethesda Md.)); and (2)
an approach based on crystallographic studies of antigen-antibody
complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)).
As used herein, a CDR may refer to CDRs defined by either approach
or by a combination of both approaches.
[0029] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0030] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra, or and Edelman, G. M. et al. (1969) Proc. Natl. Acad. Sci.
USA 63:78-85).
[0031] "Fv" as used herein may refer to the minimum antibody
fragment which contains a complete antigen-recognition and -binding
site. This fragment typically consists of a dimer of one heavy- and
one light-chain variable region domain in tight, non-covalent
association. From the folding of these two domains emanate six
hypervariable loops (3 loops each from the H and L chain) that
contribute the amino acid residues for antigen binding and confer
antigen binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three HVRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0032] A "constant region" (the term "constant domain" may be used
interchangeably herein) of an antibody refers to the constant
region of the antibody light chain (CL) or the constant region of
the antibody heavy chain (CH), either alone or in combination. A
constant region of an antibody generally provides structural
stability and other biological functions such as antibody chain
association, secretion, transplacental mobility, and complement
binding, but is not involved with binding to the antigen. The amino
acid sequence and corresponding exon sequences in the genes of the
constant region is dependent upon the species from which it is
derived; however, variations in the amino acid sequence leading to
allotypes is relatively limited for particular constant regions
within a species. The variable region of each chain is joined to
the constant region by a linking polypeptide sequence. The linkage
sequence is coded by a "J" sequence in the light chain gene, and a
combination of a "D" sequence and a "J" sequence in the heavy chain
gene. Depending on the antibody isotype, a heavy chain constant
region may include a CH1 domain, a hinge region, a CH2 domain, a
CH3 domain, and/or a CH4 domain. In certain embodiments, a heavy
chain constant region comprises a CH1 domain, a hinge region, a CH2
domain, and a CH3 domain.
[0033] The term "Fc region" (the term "Fc domain" may be used
interchangeably herein) herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native-sequence
Fc regions and variant Fc regions. The boundaries of the Fc region
of an immunoglobulin heavy chain might vary; in some embodiments,
the Fc region may include one or more amino acids of the hinge
region. In some embodiments, the human IgG heavy-chain Fc region is
defined to stretch from an amino acid residue at EU position 216 to
the carboxyl-terminus thereof. Suitable native-sequence Fc regions
for use in the antibodies of the present disclosure include human
IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
[0034] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of the sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0035] The term "diabodies" refers to antibody fragments prepared
by constructing sFv fragments (see preceding paragraph) with short
linkers (e.g., about 5-12 residues) between the V.sub.H and V.sub.L
domains such that inter-chain but not intra-chain pairing of the V
domains is achieved, thereby resulting in a bivalent fragment,
i.e., a fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described in
greater detail in, for example, EP 404,097; WO 93/11161; Hollinger
et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
[0036] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared.
[0037] As used herein, "antibody-dependent cell-mediated
cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which
nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g.,
natural killer (NK) cells, neutrophils, or macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC activity of a molecule of interest can be
assessed using an in vitro ADCC assay, such as that described in
U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
NK cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al., 1998, PNAS (USA),
95:652-656.
[0038] "Complement dependent cytotoxicity" and "CDC" refer to the
lysing of a target in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule (e.g., an
antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g., as described in Gazzano-Santoro et
al., J. Immunol. Methods, 202:163 (1996), may be performed.
[0039] The terms "polypeptide," "oligopeptide," "peptide," and
"protein" are used interchangeably herein to refer to polymers of
amino acids of any length. The polymer may be linear or branched,
it may comprise modified amino acids, and it may be interrupted by
non-amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that, because the polypeptides of the present
disclosure are based upon a tetravalent antibody, the polypeptides
can occur as single chains or associated chains.
[0040] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and/or RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imparted
before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such
as by conjugation with a labeling component. Other types of
modifications include, for example, "caps," substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, cabamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing
pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, ply-L-lysine, etc.), those
with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron,
oxidative metals, etc.), those containing alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
well as unmodified forms of the polynucleotide(s). Further, any of
the hydroxyl groups ordinarily present in the sugars may be
replaced, for example, by phosphonate groups, phosphate groups,
protected by standard protecting groups, or activated to prepare
additional linkages to additional nucleotides, or may be conjugated
to solid supports. The 5' and 3' terminal OH can be phosphorylated
or substituted with amines or organic capping group moieties of
from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized
to standard protecting groups. Polynucleotides can also contain
analogous forms of ribose or deoxyribose sugars that are generally
known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl,
2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs,
.alpha.-anomeric sugars, epimeric sugars such as arabinose,
xyloses, lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,
acyclic analogs, and abasic nucleoside analogs such as methyl
ribosides. One or more phosphodiester linkages may be replaced by
alternative linking groups. These alternative linking groups
include, but are not limited to, embodiments wherein phosphate is
replaced by P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2
("amidate"), P(O)R, P(O)OR', CO, or CH.sub.2 ("formacetal"), in
which each R or R' is independently H or substituted or
unsubstituted alkyl (1-20 C) optionally containing an ether (--O--)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not
all linkages in a polynucleotide need be identical. The preceding
description applies to all polynucleotides referred to herein,
including RNA and DNA.
[0041] As used herein, "vector" means a construct that is capable
of delivering and desirably expressing one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0042] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0043] As used herein, an "effective dosage" or "therapeutically
effective amount" of drug, compound, or pharmaceutical composition
is an amount sufficient to effect beneficial, desired, and/or
therapeutic results. For prophylactic use, beneficial or desired
results include results such as eliminating or reducing the risk,
lessening the severity, or delaying the onset of the disease,
including biochemical, histological and/or behavioral symptoms of
the disease, its complications and intermediate pathological
phenotypes presenting during development of the disease. For
therapeutic use, beneficial or desired results include clinical
results such as decreasing one or more symptoms resulting from the
disease, increasing the quality of life of those suffering from the
disease, decreasing the dose of other medications required to treat
the disease, enhancing effect of another medication such as via
targeting, delaying the progression of the disease, and/or
prolonging survival. In the case of treating an individual awaiting
a transplantation, for example, an effective amount of the drug may
reduce to some extent the level of alloantibodies and/or PRA in the
individual. In the case of treating an individual receiving a
transplantation or transfusion, an effective amount of the drug may
have the effect in and/or relieving to some extent one or more of
the symptoms or conditions (such as graft rejection) associated
with the transplantation or transfusion. An effective amount can be
administered in one or more administrations. For purposes of the
present disclosure, an effective amount of drug, compound, or
pharmaceutical composition is an amount sufficient to accomplish
prophylactic or therapeutic treatment either directly or
indirectly. An effective dosage can be administered in one or more
administrations. For purposes of the present disclosure, an
effective dosage of drug, compound, or pharmaceutical composition
is an amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0044] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during, or after
administration of the other treatment modality to the
individual.
[0045] As used herein, "treatment" or "treating" is an approach for
obtaining beneficial or desired results, including desirably
clinical results. Beneficial, desired, and/or therapeutic clinical
results include, but are not limited to, one or more of the
following: reducing or abrogating one or more symptoms of
inflammation or autoimmunity (e.g., stemming from a T-cell mediated
inflammatory disease), increasing the likelihood of a successful
patient outcome and/or mitigating one or more contraindications or
detrimental outcomes related to a medical treatment (e.g., related
to a transplantation or transfusion), decreasing symptoms resulting
from the disease, increasing the quality of life of those suffering
from the disease, decreasing the dose of other medications required
to treat the disease, delaying the progression of the disease,
and/or prolonging survival of individuals.
[0046] As used herein, "delaying development of a disease" means to
defer, hinder, slow, retard, stabilize, and/or postpone development
of the disease (such as cancer). This delay can be of varying
lengths of time, depending on the history of the disease and/or
individual being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop the disease.
For example, a symptom of an inflammatory disease, such as a T-cell
mediated inflammatory disease, may be delayed.
[0047] An "individual" or a "subject" is a mammal, more desirably a
human. Mammals also include, but are not limited to, farm animals,
sport animals, pets (such as cats, dogs, or horses), primates,
mice, and rats.
[0048] As used herein, the term "specifically recognizes" or
"specifically binds" refers to measurable and reproducible
interactions such as attraction or binding between a target and an
antibody (e.g., a full-length antibody, an antibody fragment, or an
antibody VH-VL binding unit) that is determinative of the presence
of the target in the presence of a heterogeneous population of
molecules including biological molecules. For example, an antibody,
antibody fragment, or antibody VH-VL binding unit that specifically
or preferentially binds to an epitope is an antibody that binds
this epitope with greater affinity, avidity, more readily, and/or
with greater duration than it binds to other epitopes of the target
or non-target epitopes. It is also understood by reading this
definition that, for example, an antibody, antibody fragment, or
antibody VH-VL binding unit that specifically or preferentially
binds to a first target may or may not specifically or
preferentially bind to a second target. As such, "specific binding"
or "preferential binding" does not necessarily require (although it
can include) exclusive binding. An antibody, antibody fragment, or
antibody VH-VL binding unit that specifically binds to a target may
have an association constant of greater than or about
10.sup.3M.sup.-1 or about 10.sup.4M.sup.-1, sometimes about
10.sup.5 M.sup.-1 or about 10.sup.6M.sup.-1, in other instances
about 10.sup.6M.sup.-1 or about 10.sup.7M.sup.-1, about
10.sup.8M.sup.-1 to about 10.sup.9M.sup.-1, or about 10.sup.10
M.sup.-1 to about 10.sup.11 M.sup.-1 or higher. A variety of
immunoassay formats can be used to select antibodies, antibody
fragments, or antibody VH-VL binding units that are specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See, e.g.,
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, for a description of immunoassay
formats and conditions that can be used to determine specific
immunoreactivity.
[0049] A "package insert" refers to instructions customarily
included in commercial packages of medicaments that contain
information about the indications customarily included in
commercial packages of medicaments that contain information about
the indications, usage, dosage, administration, contraindications,
other medicaments to be combined with the packaged product, and/or
warnings concerning the use of such medicaments, etc.
[0050] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise. For example, reference to an
"antibody" is a reference to from one to many antibodies, such as
molar amounts, and includes equivalents thereof known to those
skilled in the art, and so forth.
[0051] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X."
[0052] It is understood that aspect and variations of the present
disclosure described herein include "consisting" and/or "consisting
essentially of" aspects and variations.
II. Tetravalent Antibodies
[0053] Certain aspects of the present disclosure relate to
tetravalent antibodies that specifically bind to human PSGL-1. In
some embodiments, a tetravalent antibody of the present disclosure
comprises a dimer of two monomers. As described infra, the monomers
may be coupled using any means known in the art, including without
limitation wild-type interactions between antibody Fc domains or
regions, altered or mutated interactions between antibody Fc
domains or regions (e.g., using a hinge region mutation described
herein), or other artificial covalent or non-covalent interactions
(e.g., cross-linking or a linker). Exemplary tetravalent antibodies
and antibody formats are described below and illustrated in FIGS.
1A & 1B.
[0054] Human PSGL-1 may also be referred to as selectin P ligand,
SELPG, CLA, CD162, or PSGL1. In some embodiments, a tetravalent
antibody of the present disclosure binds to a polypeptide encoded
by the human SELPG gene, e.g., as described by NCBI RefSeq Gene ID
No. 6404. In some embodiments, a tetravalent antibody of the
present disclosure binds to a human PSGL-1 polypeptide containing
15 or 16 decamer repeats. In some embodiments, a tetravalent
antibody of the present disclosure binds to a polypeptide
comprising the amino acid sequence of SEQ ID NO:31. In some
embodiments, a tetravalent antibody of the present disclosure binds
to a polypeptide comprising the amino acid sequence of SEQ ID
NO:32. In some embodiments, a tetravalent antibody of the present
disclosure binds to a polypeptide comprising the amino acid
sequence of SEQ ID NO:31 and binds to a polypeptide comprising the
amino acid sequence of SEQ ID NO:32. The amino acid sequence of SEQ
ID NO:31 depicts full length human PSGL-1, GenBank.TM. accession
number AAA74577.1, GL902797, and the amino acid sequence of SEQ ID
NO:32 depicts the shorter 402 amino acid human PSGL-1 protein
(GenBank.TM. accession number XP_005269133). In specific
embodiments, a tetravalent antibody described herein specifically
binds to human PSGL-1 as determined, e.g., by ELISA or other
antigen-binding assay known in the art, or described herein.
[0055] In some embodiments, a VH domain and a VL domain of the
present disclosure form a VH-VL binding unit (e.g., that
specifically binds an epitope, such as an epitope of human PSGL-1).
As described herein, a VH-VL binding unit may be formed between a
VH domain and a VL domain using wild-type VH-VL interactions, or a
VH-VL binding unit may be further stabilized using one or more
mutations or chemical bonds (e.g., a disulfide bond, such as the
vH44-vL100 disulfide bond introduced by cysteine substitutions in
the VH and VL domain of SEQ ID NOs: 29 and 30, respectively).
[0056] In some embodiments, a tetravalent antibody of the present
disclosure comprises a dimer of two monomers, where each monomer of
the dimer comprises a single-chain polypeptide.
[0057] In some embodiments, a single-chain, heavy chain, and/or
light chain polypeptide of the present disclosure comprises a
linker sequence. A variety of linker sequences may suitably be
used, e.g., to link VH and VL domains of a VH-VL binding unit, to
link a VH or VL domain of a VH-VL binding unit to a VH or VL domain
of another VH-VL binding unit, or to link a VH or VL domain of a
VH-VL binding unit to an antibody constant region, such as an Fc
domain or region. In some embodiments, a linker of the present
disclosure may be present between domains or regions. In some
embodiments, two domains or regions of the present disclosure may
be joined without a linker, or the linker joining two domains or
regions may be removed. Coupling of such single-chain fragments
using various linkers is described in Kortt et al., 1997, Protein
Engineering, 10:423-433. In some embodiments, a linker sequence of
the present disclosure comprises 1-50 amino acids. In certain
embodiments, a linker sequence of the present disclosure comprises
5-12 amino acids. Exemplary linker sequences are described herein
and illustrated in FIG. 1A. In some embodiments, a linker sequence
of the present disclosure comprises one or more repeats of the
amino acid sequence of GGGGS (SEQ ID NO:25). In some embodiments, a
linker sequence of the present disclosure comprises two, three,
four, or five repeats of the amino acid sequence of GGGGS (SEQ ID
NO:25). In some embodiments, a linker sequence of the present
disclosure comprises the amino acid sequence of SEQ ID NO:33, 34,
35, or 36. In some embodiments, a linker sequence of the present
disclosure comprises the amino acid sequence of GGGGSAAA (SEQ ID
NO:26). In some embodiments, a linker sequence of the present
disclosure comprises the amino acid sequence of ASTGS (SEQ ID
NO:27). In some embodiments, a linker sequence of the present
disclosure comprises the amino acid sequence of ASTGSGGGGS (SEQ ID
NO:28).
[0058] In some embodiments, a tetravalent antibody of the present
disclosure comprises a dimer of two single-chain diabodies (scDbs),
which may optionally be fused to an antibody constant region, such
as an Fc domain.
[0059] In some embodiments, each monomer of the dimer comprises a
single-chain polypeptide comprising (a) two light chain variable
(VL) domains, wherein each of the two VL domains comprises a
CDR-L1, a CDR-L2, and a CDR-L3, and wherein the two VL domains are
specific for human PSGL-1; (b) two heavy chain variable (VH)
domains, wherein each of the two VH domains comprises a CDR-H1, a
CDR-H2, and a CDR-H3, and wherein the two VH domains are specific
for human PSGL-1; and (c) an antibody Fc domain. In some
embodiments, each of the two VL domains forms a VH-VL binding unit
with a corresponding VH domain of the two VH domains.
[0060] In certain embodiments, each of the two single-chain
polypeptides comprises, from N-terminus to C-terminus: (a) a first
VL domain of two VL domains; (b) a first linker sequence; (c) a
first VH domain of two VH domains; (d) a second linker sequence;
(e) a second VL domain of two VL domains; (f) a third linker
sequence; (g) a second VH domain of two VH domains; (h) a fourth
linker sequence; and (i) an antibody Fc domain. In some
embodiments, the first VL domain forms a VH-VL binding unit with
the second VH domain, and the first VH domain forms a VH-VL binding
unit with the second VL domain.
[0061] In some embodiments, the first, second and third linker
sequences each comprise two or more repeats of the amino acid
sequence of SEQ ID NO:25. In some embodiments, the first, second or
third linker sequence comprises the amino acid sequence of SEQ ID
NO:33, 34, 35, or 36. In some embodiments, the first and the third
linker sequences have the same sequence and comprise two repeats of
SEQ ID NO:25. In some embodiments, the second linker sequence
comprises five repeats of SEQ ID NO:25. In some embodiments, the
fourth linker sequence comprises the amino acid sequence of SEQ ID
NO:26.
[0062] In some embodiments, a tetravalent antibody of the present
disclosure comprises a dimer of two tandem single-chain variable
fragment (scFv) units (termed taFv for tandem scFv), which may
optionally be fused to an antibody constant domain, such as an Fc
domain of a heavy chain constant domain.
[0063] In certain embodiments, each of the two single-chain
polypeptides comprises, from N-terminus to C-terminus: (a) a first
VH domain of the two VH domains; (b) a first linker sequence; (c) a
first VL domain of the two VL domains; (d) a second linker
sequence; (e) a second VL domain of the two VL domains; (f) a third
linker sequence; (g) a second VH domain of the two VH domains; (h)
a fourth linker sequence; and (i) an antibody Fc domain. In some
embodiments, the first VL domain forms a VH-VL binding unit with
the first VH domain, and the second VH domain forms a VH-VL binding
unit with the second VL domain. In other embodiments, each of the
two single-chain polypeptides comprises, from N-terminus to
C-terminus: (a) a first VL domain of the two VL domains; (b) a
first linker sequence; (c) a first VH domain of the two VH domains;
(d) a second linker sequence; (e) a second VH domain of the two VH
domains; (f) a third linker sequence; (g) a second VL domain of the
two VL domains; (h) a fourth linker sequence; and (i) the heavy
chain constant domain comprising an antibody Fc domain.
[0064] In some embodiments, the first and the third linker
sequences have the same sequence comprising five repeats of SEQ ID
NO:25. In some embodiments, the second linker sequence comprises
the amino acid sequence of SEQ ID NO:27. In some embodiments, the
fourth linker sequence comprises the amino acid sequence of SEQ ID
NO:26.
[0065] In some embodiments, a tetravalent antibody of the present
disclosure comprises a dimer of two monomers, where each monomer of
the dimer comprises an antibody heavy chain and an antibody light
chain.
[0066] In some embodiments, a tetravalent antibody of the present
disclosure comprises a light chain comprising (i) two light chain
variable (VL) domains, wherein each of the two VL domains comprises
a CDR-L1, a CDR-L2, and a CDR-L3, and wherein the two VL domains
are specific for human PSGL-1, (ii) a first heavy chain variable
(VH) domain, and (iii) a light chain constant (CL) domain; and/or a
heavy chain comprising (i) a second heavy chain variable (VH)
domain, and (ii) a heavy chain constant region comprising a first
heavy chain constant region (CH1) domain, an antibody hinge region,
an second heavy chain constant region (CH2) domain, and a third
heavy chain constant region (CH3) domain. In some embodiments, the
first and the second VH domains each comprise a CDR-H1, a CDR-H2,
and a CDR-H3. In some embodiments, the first and the second VH
domains are specific for human PSGL-1. In some embodiments, each of
the two VL domains forms a VH-VL binding unit with a corresponding
VH domain of the first and the second VH domains.
[0067] In certain embodiments, the antibody light chain comprises,
from N-terminus to C-terminus: (a) the first VH domain; (b) a first
linker sequence; (c) a first VL domain of the two or more VL
domains; (d) a second linker sequence; (e) a second VL domain of
the two or more VL domains; and (f) the CL domain. In some
embodiments, the CL domain is a kappa CL domain. In other
embodiments, the CL domain is a lambda CL domain. In some
embodiments, the first VL domain forms a VH-VL binding unit with
the first VH domain, and the second VH domain forms a VH-VL binding
unit with the second VL domain.
[0068] In some embodiments, the first linker sequence comprises
five repeats of SEQ ID NO:25. In some embodiments, the second
linker sequence comprises the amino acid sequence of SEQ ID
NO:28.
[0069] In some embodiments, a tetravalent antibody of the present
disclosure comprises a light chain comprising, from N-terminus to
C-terminus: (a) a first VL domain of the two VL domains; (b) the CL
domain; (c) a first linker sequence; (d) the first VH domain; (e) a
second linker sequence; and (f) a second VL domain of the two VL
domains. In some embodiments, the CL domain is a kappa CL domain.
In other embodiments, the CL domain is a lambda CL domain.
[0070] In some embodiments, the first linker sequence comprises two
repeats of SEQ ID NO:25. In some embodiments, the second linker
sequence comprises five repeats of SEQ ID NO:25.
[0071] In some embodiments, a tetravalent antibody of the present
disclosure comprises a heavy chain comprising, from N-terminus to
C-terminus: (a) the second of two VH domains; and (b) a heavy chain
constant region comprising a first heavy chain constant region
(CH1) domain, an antibody hinge region, an second heavy chain
constant region (CH2) domain, and a third heavy chain constant
region (CH3) domain. In some embodiments, the antibody Fc domain
comprises a heavy chain constant 2 (CH2) domain and a heavy chain
constant 3 (CH3) domain. In some embodiments, the first VL domain
forms a VH-VL binding unit with the first VH domain, and the second
VH domain forms a VH-VL binding unit with the second VL domain.
[0072] In some embodiments, a tetravalent antibody of the present
disclosure comprises a light chain comprising (i) a first heavy
chain variable (VH) domain, (ii) a first light chain variable (VL)
domain, and (iii) a light chain constant (CL) domain; and/or a
heavy chain comprising (i) a second heavy chain variable (VH)
domain, (ii) a second light chain variable (VL) domain, and (iii) a
heavy chain constant region comprising a first heavy chain constant
region (CH1) domain, an antibody hinge region, an second heavy
chain constant region (CH2) domain, and a third heavy chain
constant region (CH3) domain. In some embodiments, each of the
first and second VL domains comprises a CDR-L1, a CDR-L2, and a
CDR-L3. In some embodiments, the first and second VL domains are
specific for human PSGL-1. In some embodiments, each of the first
and second VH domains comprises a CDR-H1, a CDR-H2, and a CDR-H3.
In some embodiments, the first and second VH domains are specific
for human PSGL-1. In some embodiments, each of the first and second
VL domains forms a VH-VL binding unit with a corresponding VH
domain of the first and second VH domains.
[0073] In some embodiments, the antibody light chain comprises,
from N-terminus to C-terminus: (a) the first VH domain; (b) a first
linker sequence; (c) the first VL domain; and (d) the CL domain. In
some embodiments, the CL domain is a kappa CL domain. In other
embodiments, the CL domain is a lambda CL domain.
[0074] In some embodiments, the first linker sequence comprises
five repeats of SEQ ID NO:25.
[0075] In some embodiments, the antibody heavy chain comprises,
from N-terminus to C-terminus: (a) the second of two VH domains;
(b) a second linker sequence; (c) the second of two VL domains; and
(d) a heavy chain constant region comprising a first heavy chain
constant region (CH1) domain, an antibody hinge region, an second
heavy chain constant region (CH2) domain, and a third heavy chain
constant region (CH3) domain. In some embodiments, the antibody Fc
domain comprises a heavy chain constant 2 (CH2) domain and a heavy
chain constant 3 (CH3) domain.
[0076] In some embodiments, the second linker sequence comprises
five repeats of SEQ ID NO:25.
[0077] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VH domains comprising one or more
CDRs selected from (i) a CDR-H1 comprising the amino acid sequence
of SFGMH (SEQ ID NO:17); (ii) a CDR-H2 comprising the amino acid
sequence of YINGGSSTIFYANAVKG (SEQ ID NO:18); and (iii) a CDR-H3
comprising the amino acid sequence of YASYGGGAMDY (SEQ ID NO:19).
In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VH domains comprising (i) a CDR-H1
comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2
comprising the amino acid sequence of SEQ ID NO:18; and (iii) a
CDR-H3 comprising the amino acid sequence of SEQ ID NO:19. In some
embodiments, a tetravalent antibody of the present disclosure is a
dimer of two monomers, each monomer comprising two VH domains, each
VH domain comprising one or more CDRs selected from (i) a CDR-H1
comprising the amino acid sequence of SFGMH (SEQ ID NO:17); (ii) a
CDR-H2 comprising the amino acid sequence of YINGGSSTIFYANAVKG (SEQ
ID NO:18); and (iii) a CDR-H3 comprising the amino acid sequence of
YASYGGGAMDY (SEQ ID NO:19). In some embodiments, a tetravalent
antibody of the present disclosure is a dimer of two monomers, each
monomer comprising two VH domains, each VH domain comprising (i) a
CDR-H1 comprising the amino acid sequence of SEQ ID NO:17; (ii) a
CDR-H2 comprising the amino acid sequence of SEQ ID NO:18; and
(iii) a CDR-H3 comprising the amino acid sequence of SEQ ID
NO:19.
[0078] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VH domains comprising the amino
acid sequence of SEQ ID NO:23. In some embodiments, a tetravalent
antibody of the present disclosure comprises a monomer comprising
two VH domains, each VH domain comprising the amino acid sequence
of SEQ ID NO:23. In some embodiments, a tetravalent antibody of the
present disclosure comprises one or more VH domains comprising the
amino acid sequence of SEQ ID NO:29. In some embodiments, a
tetravalent antibody of the present disclosure comprises a monomer
comprising two VH domains, each VH domain comprising the amino acid
sequence of SEQ ID NO:29.
[0079] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VH domains comprising a sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:23. In some embodiments, a tetravalent antibody of the present
disclosure comprises a monomer comprising two VH domains, each VH
domain comprising a sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:23. In some embodiments, the VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human PSGL-1 antibody comprising that
sequence retains the ability to bind to human PSGL-1. In some
embodiments, total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:23.
[0080] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VH domains comprising a sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:29. In some embodiments, a tetravalent antibody of the present
disclosure comprises a monomer comprising two VH domains, each VH
domain comprising a sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:29. In some embodiments, the VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human PSGL-1 antibody comprising that
sequence retains the ability to bind to human PSGL-1. In some
embodiments, total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:29.
[0081] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VL domains comprising one or more
CDRs selected from (i) a CDR-L1 comprising the amino acid sequence
of RSSQSIVHNDGNTYFE (SEQ ID NO:20); (ii) a CDR-L2 comprising the
amino acid sequence of KVSNRFS (SEQ ID NO:21); and (iii) a CDR-L3
comprising the amino acid sequence of FQGSYVPLT (SEQ ID NO:22). In
some embodiments, a tetravalent antibody of the present disclosure
comprises one or more VL domains comprising (i) a CDR-L1 comprising
the amino acid sequence of RSSQSIVHNDGNTYFE (SEQ ID NO:20); (ii) a
CDR-L2 comprising the amino acid sequence of KVSNRFS (SEQ ID
NO:21); and (iii) a CDR-L3 comprising the amino acid sequence of
FQGSYVPLT (SEQ ID NO:22). In some embodiments, a tetravalent
antibody of the present disclosure is a dimer of two monomers, each
monomer comprising two VL domains, each VL domain comprising one or
more CDRs selected from (i) a CDR-L1 comprising the amino acid
sequence of RSSQSIVHNDGNTYFE (SEQ ID NO:20); (ii) a CDR-L2
comprising the amino acid sequence of KVSNRFS (SEQ ID NO:21); and
(iii) a CDR-L3 comprising the amino acid sequence of FQGSYVPLT (SEQ
ID NO:22). In some embodiments, a tetravalent antibody of the
present disclosure is a dimer of two monomers, each monomer
comprising two VL domains, each VL domain comprising (i) a CDR-L1
comprising the amino acid sequence of RSSQSIVHNDGNTYFE (SEQ ID
NO:20); (ii) a CDR-L2 comprising the amino acid sequence of KVSNRFS
(SEQ ID NO:21); and (iii) a CDR-L3 comprising the amino acid
sequence of FQGSYVPLT (SEQ ID NO:22).
[0082] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VL domains comprising the amino
acid sequence of SEQ ID NO:24. In some embodiments, a tetravalent
antibody of the present disclosure comprises a monomer comprising
two VL domains, each VL domain comprising the amino acid sequence
of SEQ ID NO:24. In some embodiments, a tetravalent antibody of the
present disclosure comprises one or more VL domains comprising the
amino acid sequence of SEQ ID NO:30. In some embodiments, a
tetravalent antibody of the present disclosure comprises a monomer
comprising two VL domains, each VL domain comprising the amino acid
sequence of SEQ ID NO:30.
[0083] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VL domains comprising a sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:24. In some embodiments, a tetravalent antibody of the present
disclosure comprises a monomer comprising two VL domains, each VL
domain comprising a sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:24. In some embodiments, the VL
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human PSGL-1 antibody comprising that
sequence retains the ability to bind to human PSGL-1. In some
embodiments, total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:24.
[0084] In some embodiments, a tetravalent antibody of the present
disclosure comprises one or more VL domains comprising a sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:30. In some embodiments, a tetravalent antibody of the present
disclosure comprises a monomer comprising two VL domains, each VL
domain comprising a sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:30. In some embodiments, the VL
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human PSGL-1 antibody comprising that
sequence retains the ability to bind to human PSGL-1. In some
embodiments, total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:30.
[0085] In some embodiments, a tetravalent antibody of the present
disclosure comprises a single-chain polypeptide comprising a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NOs:1, 3, or 5. In some embodiments, a tetravalent antibody
of the present disclosure comprises a dimer of two monomers, each
monomer comprising two single-chain polypeptides, each single-chain
polypeptide comprising a sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NOs:1, 3, or 5. In some embodiments,
the single-chain polypeptide sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
PSGL-1 antibody comprising that sequence retains the ability to
bind to human PSGL-1. In some embodiments, total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NOs:1, 3, or 5. In some embodiments, a tetravalent antibody of the
present disclosure comprises two single-chain polypeptides, each
comprising the amino acid sequence of SEQ ID NOs:1, 3, or 5.
[0086] In some embodiments, a tetravalent antibody of the present
disclosure comprises a single-chain polypeptide comprising a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to a single-chain polypeptide
encoded by the polynucleotide sequence of SEQ ID NOs:2, 4, or 6. In
some embodiments, a tetravalent antibody of the present disclosure
comprises a dimer of two monomers, each monomer comprising two
single-chain polypeptides, each single-chain polypeptide comprising
a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to a single-chain polypeptide
encoded by the polynucleotide sequence of SEQ ID NOs:2, 4, or 6. In
some embodiments, the single-chain polypeptide sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human PSGL-1 antibody comprising that sequence retains the
ability to bind to human PSGL-1. In some embodiments, total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
the single-chain polypeptide encoded by the polynucleotide sequence
of SEQ ID NOs:2, 4, or 6. In some embodiments, a tetravalent
antibody of the present disclosure comprises two single-chain
polypeptides, each encoded by the polynucleotide sequence of SEQ ID
NOs:2, 4, or 6.
[0087] In some embodiments, a tetravalent antibody of the present
disclosure comprises a light chain polypeptide comprising a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NOs:7, 9, or 13. In some embodiments, a tetravalent antibody
of the present disclosure comprises a dimer of two monomers, each
monomer comprising a heavy chain and a light chain, and each light
chain comprising a sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NOs:7, 9, or 13. In some embodiments,
the light chain polypeptide sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
PSGL-1 antibody comprising that sequence retains the ability to
bind to human PSGL-1. In some embodiments, total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NOs:7, 9, or 13. In some embodiments, a tetravalent antibody of the
present disclosure comprises a dimer of two monomers, each monomer
comprising a heavy chain and a light chain, and each light chain
comprising the amino acid sequence of SEQ ID NOs:7, 9, or 13.
[0088] In some embodiments, a tetravalent antibody of the present
disclosure comprises a light chain polypeptide comprising a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to a light chain polypeptide
encoded by the polynucleotide sequence of SEQ ID NOs:8, 10, or 14.
In some embodiments, a tetravalent antibody of the present
disclosure comprises a dimer of two monomers, each monomer
comprising a heavy chain and a light chain, and each light chain
comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to a light chain
polypeptide encoded by the polynucleotide sequence of SEQ ID NOs:8,
10, or 14. In some embodiments, the light chain polypeptide
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human PSGL-1 antibody comprising that
sequence retains the ability to bind to human PSGL-1. In some
embodiments, total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in the light chain polypeptide encoded by
the polynucleotide sequence of SEQ ID NOs:8, 10, or 14. In some
embodiments, a tetravalent antibody of the present disclosure
comprises a dimer of two monomers, each monomer comprising a heavy
chain and a light chain, and each light chain comprising a light
chain polypeptide encoded by the polynucleotide sequence of SEQ ID
NOs:8, 10, or 14.
[0089] In some embodiments, a tetravalent antibody of the present
disclosure comprises a heavy chain polypeptide comprising a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NOs:11 or 15. In some embodiments, a tetravalent antibody of
the present disclosure comprises a dimer of two monomers, each
monomer comprising a heavy chain and a light chain, and each heavy
chain comprising a sequence having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NOs:11 or 15. In some embodiments,
the heavy chain polypeptide sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
PSGL-1 antibody comprising that sequence retains the ability to
bind to human PSGL-1. In some embodiments, total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NOs:11 or 15. In some embodiments, a tetravalent antibody of the
present disclosure comprises a dimer of two monomers, each monomer
comprising a heavy chain and a light chain, and each heavy chain
comprising the amino acid sequence of SEQ ID NOs:11 or 15.
[0090] In some embodiments, a tetravalent antibody of the present
disclosure comprises a heavy chain polypeptide comprising a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to a heavy chain polypeptide
encoded by the polynucleotide sequence of SEQ ID NOs:12 or 16. In
some embodiments, a tetravalent antibody of the present disclosure
comprises a dimer of two monomers, each monomer comprising a heavy
chain and a light chain, and each heavy chain comprising a sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to a heavy chain polypeptide encoded by
the polynucleotide sequence of SEQ ID NOs:12 or 16. In some
embodiments, the heavy chain polypeptide sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human PSGL-1 antibody comprising that sequence retains the
ability to bind to human PSGL-1. In some embodiments, total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
the heavy chain polypeptide encoded by the polynucleotide sequence
of SEQ ID NOs:12 or 16. In some embodiments, a tetravalent antibody
of the present disclosure comprises a dimer of two monomers, each
monomer comprising a heavy chain and a light chain, and each heavy
chain comprising a light chain polypeptide encoded by the
polynucleotide sequence of SEQ ID NOs: 12 or 16.
[0091] The present disclosure encompasses modifications to
antibodies or polypeptide described herein, including functionally
equivalent antibodies which do not significantly affect their
properties and variants which have enhanced or decreased activity
and/or affinity. Modification of polypeptides is routine practice
in the art and need not be described in detail herein. Examples of
modified polypeptides include polypeptides with conservative
substitutions of amino acid residues, one or more deletions or
additions of amino acids which do not significantly deleteriously
change the functional activity, or use of chemical analogs.
[0092] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the serum half-life of the
antibody.
[0093] Substitution variants have at least one amino acid residue
in the antibody molecule removed and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in the
table below under the heading of "conservative substitutions." If
such substitutions result in a change in biological activity, then
more substantial changes, denominated "exemplary substitutions" in
the table below, or as further described below in reference to
amino acid classes, may be introduced and the products
screened.
Amino Acid Substitutions
TABLE-US-00002 [0094] Original Conservative Exemplary Residue
Substitutions Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys
Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;
Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln
Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu;
Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val;
Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile
Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr
Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser
Val (V) Leu Ile; Leu; Met; Phe; Ala; Norleucine
[0095] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: [0096]
(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; [0097] (2)
Polar without charge: Cys, Ser, Thr, Asn, Gln; [0098] (3) Acidic
(negatively charged): Asp, Glu; [0099] (4) Basic (positively
charged): Lys, Arg; [0100] (5) Residues that influence chain
orientation: Gly, Pro; and [0101] (6) Aromatic: Trp, Tyr, Phe,
His
[0102] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class.
[0103] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an antibody fragment such as an Fv fragment.
Exemplary cysteine mutations are described herein (e.g., the G44C
VH domain mutation of SEQ ID NO:29, or the Q100C VL domain mutation
of SEQ ID NO:30).
[0104] In some embodiments, a tetravalent antibody of the present
disclosure comprises an antibody Fc domain. In some embodiments,
the antibody Fc domain is a human Fc domain. In certain
embodiments, the antibody Fc domain is a human IgG4 Fc domain.
[0105] In some embodiments, one or more amino acid residues in the
heavy chain constant region and/or the light chain constant region
of the antibody are modified. For example, amino acid residues of
antibodies described in the Examples may be modified. In some
embodiments, the Fc region of antibodies is modified to enhance or
reduce ADCC and/or CDC activities of the antibodies. See Shields et
al., J. Biol. Chem. 276:6591-6604 (2001); Presta et al., Biochem.
Soc. Trans. 30:487-490 (2002).
[0106] In some embodiments, the Fc region of antibodies is modified
to enhance dimer formation and/or stability, or to reduce dimer
heterogeneity (e.g., shuffling). It has been demonstrated that a
Serine to Proline mutation at position 241 using Kabat numbering
(Kabat et al. 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) or at position 228 using the
EU index (Edelman et al, 1969, Proc. Natl. Acad. Sci. USA, 63(1):
78-85) in the hinge region of human IgG4 results in considerable
reduction of intra-chain disulfide bond formation, resulting in the
reduction of IgG4 "half-antibody" molecules and reduced
heterogeneity/shuffling of IgG4 molecules (Bloom et al. 1997,
Protein Sci, 6:407-415; Angal et al, 1993, Molecular Immunology,
30(1): 105-108)). There are also published reports that this hinge
mutation may decrease IgG4 shuffling and increase the half-life of
the IgG4 molecules in vivo (Labrijn, et al, 2009, Nat Biotechnol
27:767-771; Stubenrauch, et al, 2010, Drug Metab Dispos 38:84-91).
Van der Neut Kolfschoten et al, reported that the C.sub.H3 domain
of IgG4 and not the core hinge is predominantly involved in the Fab
arm exchange reaction (see Van der Neut Kolfschoten et al, 2007,
Science, 317: 1554-1557 ("Van der Neut Kolfschoten") at page 1555,
col. 2). Van der Neut Kolfschaten reported that exchanging the
C.sub.H3 domain of IgG1 for the C.sub.H3 domain of IgG4 activated
Fab arm exchange for the IgG1, while exchanging the C.sub.H3 domain
of IgG4 abrogated Fab arm exchange for the IgG4 (see, p. 1555 and
FIG. 2D).
[0107] In a specific embodiment, provided herein are tetravalent
antibodies, that specifically bind to PSGL-1, and that contain one
or more amino acid substitutions in the IgG4 hinge region, wherein
said antibody or antigen-binding fragment thereof retains specific
binding to said PSGL-1 and wherein IgG4 shuffling is reduced
relative to an antibody comprising an IgG4 hinge region not
comprising said one or more amino acid substitutions. In a specific
embodiment, the IgG4 hinge region only comprises a single amino
acid substitution. An example of a "human IgG4 hinge region," is
the region on the heavy chain of an IgG4 antibody between the
C.sub.HI and C.sub.H2 domains, as set forth in Angal et al., 1993,
Molecular Immunology, 30(1): 105-108.
[0108] In a specific embodiment, a reduction in IgG4 shuffling is
determined by detecting of a lower amount of half antibody
molecules or of arm exchange produced from an antibody described
herein which contains one or more amino acid substitutions in the
hinge region, as compared to the amount of half antibody molecules
or of arm exchange produced from an IgG4 molecule containing an
IgG4 hinge region not comprising said one or more amino acid
substitutions. Any assay well-known in the art can be used to
detect half antibody production and bispecific antibody molecules.
See, e.g., Van der Neut Kolfschoten et al, 2007, Science, 317:
1554-1557, for examples of assays to detect production of
bispecific antibodies.
[0109] In a specific embodiment, provided herein are tetravalent
antibodies that specifically bind to PSGL-1 and include a human
IgG4 Fc domain comprising a Serine to Proline amino acid
substitution at amino acid position 228 of the heavy chain numbered
according to the EU index (also known as position 241 using Kabat
numbering).
[0110] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Hefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect antibody-dependent cellular cytotoxicity (ADCC). In
particular, CHO cells with tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., 1999, Mature
Biotech. 17:176-180).
[0111] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0112] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0113] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g., antibodies, as potential therapeutics is
rarely the native cell, variations in the glycosylation pattern of
the antibodies can be expected (see, e.g., Hse et al., 1997, J.
Biol. Chem. 272:9062-9070).
[0114] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes, and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261; and 5,278,299). Glycosylation, or
certain types of glycosylation, can be enzymatically removed from
the glycoprotein, for example using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2, or
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0115] In some embodiments, an antibody of the present disclosure
is modified using coupling techniques known in the art, including,
but not limited to, enzymatic means, oxidative substitution, and
chelation. Modifications can be used, for example, for attachment
of labels for immunoassay.
[0116] The tetravalent antibody or polypeptide of the present
disclosure may be conjugated (for example, linked) to an agent,
such as a therapeutic agent or a label. Examples of therapeutic
agents are radioactive moieties, cytotoxins, and chemotherapeutic
molecules.
[0117] The tetravalent antibody (or polypeptide) of the present
disclosure may be linked to a label such as a fluorescent molecule,
a radioactive molecule, an enzyme, or any other labels known in the
art. As used herein, the term "label" refers to any molecule that
can be detected.
[0118] In a certain embodiment, an antibody may be labeled by
incorporation of a radiolabeled amino acid. In a certain
embodiment, biotin moieties that can be detected by marked avidin
(e.g., streptavidin containing a fluorescent marker or enzymatic
activity that can be detected by optical or colorimetric methods)
may be attached to the antibody. In certain embodiments, a label
may be incorporated into or attached to another reagent which in
turn binds to the antibody of interest. For example, a label may be
incorporated into or attached to an antibody that in turn
specifically binds the antibody of interest. In certain
embodiments, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Certain general classes of labels include, but
are not limited to, enzymatic, fluorescent, chemiluminescent, and
radioactive labels. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionucleoides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I or .sup.131I)
fluorescent labels (e.g., fluorescein isothocyanate (FITC),
rhodamine, lanthanide phosphors, or phycoerythrin (PE)), enzymatic
labels (e.g., horseradish peroxidase, .beta.-galactosidase,
luciferase, alkaline phosphatase, glucose oxidase,
glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate
dehydrogenase, penicillinase, or luciferase), chemiluminescent,
biotinyl groups, predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, or epitope
tags). In certain embodiments, labels are attached by spacer arms
of various lengths to reduce potential steric hindrance.
[0119] Based on the description herein, a tetravalent antibody of
the present disclosure may be tested according to a variety of in
vitro and in vivo assays known in the art. Such assays may include,
e.g., binding assays directed to the ability of a tetravalent
antibody or fragment thereof to bind an epitope or polypeptide of
interest (e.g., human PSGL-1 or an epitope thereof), or functional
assays directed to one or more functional properties of a
tetravalent antibody or fragment thereof.
[0120] In some embodiments, a tetravalent antibody of the present
disclosure may be tested for binding activity against human PSGL-1.
In some embodiments, binding of a tetravalent antibody to human
PSGL-1 or an epitope thereof may be tested in an in vitro binding
assay. A variety of binding assays are known in the art. Such
binding assays may be cell-based assays (e.g., testing the ability
of a tetravalent antibody to bind a cell expressing human PSGL-1 or
an epitope thereof), or they may be polypeptide-based (e.g.,
testing the ability of a tetravalent antibody to bind human PSGL-1
or an epitope thereof). In some embodiments, a tetravalent antibody
of the present disclosure is tested for binding to a cell
expressing human PSGL-1 (e.g., an Sp2 cell, as exemplified infra)
by flow cytometry, FRET, histochemical assays, and the like. Other
suitable binding assays may include without limitation equilibrium
methods (e.g., enzyme-linked immunoabsorbent assay (ELISA),
radioimmunoassay (MA), Biacore.TM. analysis, indirect binding
assay, competitive inhibition assay, fluorescence resonance energy
transfer (FRET), immunoprecipitation, gel electrophoresis and
chromatography (e.g., gel filtration).
[0121] In some embodiments, a tetravalent antibody of the present
disclosure may be tested for one or more functional assays for
PSGL-1 function. In some embodiments, a tetravalent antibody of the
present disclosure may be tested for induction of apoptosis in
cell(s) expressing human PSGL-1. In some embodiments, a tetravalent
antibody of the present disclosure displays enhanced induction of
apoptosis in a target cell (e.g., a cell expressing human PSGL-1 or
an epitope thereof) as compared to a conventional (e.g., bivalent)
antibody having one or more VH or VL domains in common with the
tetravalent antibody (e.g., a parental antibody). As demonstrated
herein, tetravalent antibodies of the present disclosure displayed
greater potency in inducing apoptosis in target cells than parental
antibodies having a common VH and/or VL domain. Apoptosis assays
are described in the art and can be readily carried out by one of
skill in the art (see, e.g., Muppidi, J., Porter, M. and Siegel, R.
M. 2004. Measurement of Apoptosis and Other Forms of Cell Death.
Current Protocols in Immunology. 59:3.17.1-3.17.36). Selected
assays for detecting apoptosis (e.g., Annexin V or propidium iodide
staining) are exemplified supra. The term "induce" or "inducing"
means initiation of or an increase of apoptosis above a control
level. Apoptosis of activated T cells can be induced by about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about 90%, about 100%, about 125%, about 150% or more
compared to a control (e.g. Apoptosis of activated T cells in the
absence of the antibodies describe herein or in the presence of a
non-specific antibody).
[0122] T cells and T cell lines which are appropriate for use in
the assays described herein relating to PSGL-1 activity are readily
available (e.g., ARR, DU.528, Jurkat, H-SB2, RPMI 8402, CML-T1,
Karpas 45, KE-37/SKW-3, SUP-T1, SUP-T3, MOLT 3/4, P12-Ichikawa,
PF-382, CCRF-CEM, HPB-ALL, K-Tl, TALL-1, MOLT 16/17, TALL-104,
DND-41, Loucy, MOLT 13, Peer/Bel3, HUT 78/H9, HUT 102, MT-1, DEL,
JB6, Karpas 299, SU-DHL1, 12H5, 3D054.8, 3DO11.10, 8D051.15, or
3D018.3) or can be readily identified using methods known in the
art (see, e.g., Thornton, A. M. 2003. Fractionation of T and B
Cells Using Magnetic Beads. Current Protocols in Immunology.
55:3.5A. 1-3.5A. i 1 Hathcock, K. 2001. T Cell Enrichment by
Cytotoxic Elimination of B Cells and Accessory Cells. Current
Protocols in Immunology. 00:3.3.1-3.3.5., Horgan, K., Shaw, S. and
Boirivant, M. 2009. Immunomagnetic Purification of T Cell
Subpopulations. Current Protocols in Immunology. 85:7.4.1-7.4.9.,
and Kanof, M. E. 2001. Purification of T Cell Subpopulations.
Current Protocols in Immunology. 00:7.3.1-7.3.5). In particular
embodiments, cells or cell lines for use in cell proliferation
assays can express PSGL-1, endogenously or recombinantly. Cells or
cell lines for use in cell viability assays can express PSGL-1,
endogenously or recombinantly, and exert changes in cell viability
in response to PSGL-1 ligand or anti-PSGL-1 antibody binding. Cells
or cell lines for use in apoptosis assays can express PSGL-1,
endogenously or recombinantly, and exert changes in apoptosis in
response to PSGL-1 ligand or anti-PSGL-1 antibody binding.
Preferably the cells or cell lines are human (e.g. ARR, DU.528,
Jurkat, H-SB2, RPMI 8402, CML-Tl, Karpas 45, KE-37/SKW-3, SUP-Tl,
SUP-T3, MOLT 3/4, P12-Ichikawa, PF-382, CCRF-CEM, HPB-ALL, K-Tl,
TALL-1, MOLT 16/17, TALL-104, DND-41, Loucy, MOLT 13, Peer/Bel3,
HUT 78/H9, HUT 102, MT-1, DEL, JB6, Karpas 299, or SU-DHL1).
[0123] In some embodiments, a tetravalent antibody of the present
disclosure may be tested for inhibition of delayed type
hypersensitivity (DTH). In some embodiments, a tetravalent antibody
of the present disclosure displays enhanced inhibition of DTH
(e.g., in a trans vivo animal model) as compared to a conventional
(e.g., bivalent) antibody having one or more VH or VL domains in
common with the tetravalent antibody (e.g., a parental antibody).
As demonstrated herein, tetravalent antibodies of the present
disclosure displayed greater potency in inhibiting DTH in a trans
vivo mouse footpad swelling model than parental antibodies having a
common VH and/or VL domain. DTH assays are described in the art and
exemplified infra and can be readily carried out by one of skill in
the art. In some embodiments, a tetravalent antibody of the present
disclosure may display a potency of DTH inhibition that may be
increased by about 10%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, about 100%, about 125%,
about 150%, about 200%, about 300%, about 400%, about 500%, about
600%, or more compared to a control (e.g. inhibition of DTH by a
conventional or bivalent antibody, such as the parental
antibody).
III. Polynucleotides, Vectors, Host Cells, and Antibody
Production
[0124] The present disclosure also provides polynucleotides
comprising a polynucleotide encoding any of the tetravalent
antibodies and/or polypeptides described herein. In some
embodiments, the polypeptides comprise the sequences of light chain
and heavy chain variable regions. In some embodiments, the
polynucleotide is an isolated polynucleotide (e.g., isolated from a
host cell or from one or more different polynucleotides).
[0125] Provided herein are polynucleotides encoding any of the
tetravalent antibodies or polypeptide constituents (e.g., monomers
such as single-chain polypeptides, antibody heavy chains, and/or
antibody light chains) described herein. In some embodiments, a
polynucleotide of the present disclosure encodes a polypeptide
sequence selected from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and
17-31. In some embodiments, a polynucleotide of the present
disclosure comprises a polynucleotide sequence selected from SEQ ID
NOs:2, 4, 6, 8, 10, 12, 14, and 16. In some embodiments, a
polynucleotide of the present disclosure comprises one or more
introns. In other embodiments, a polynucleotide of the present
disclosure does not comprise an intron, e.g., a cDNA or processed
mRNA sequence.
[0126] It is appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Thus, polynucleotides that
vary due to differences in codon usage are specifically
contemplated by the present disclosure. Further, alleles of the
genes comprising the polynucleotide sequences provided herein are
within the scope of the present disclosure. Alleles are endogenous
genes that are altered as a result of one or more mutations, such
as deletions, additions, and/or substitutions of nucleotides. The
resulting mRNA and protein can, but need not, have an altered
structure or function. Alleles can be identified using standard
techniques (such as hybridization, amplification, and/or database
sequence comparison).
[0127] Also provided herein are polynucleotides that are optimized,
e.g., by codon/RNA optimization, replacement with heterologous
signal sequences, and elimination of mRNA instability elements.
Methods to generate optimized nucleic acids encoding a tetravalent
antibody or polypeptide thereof for recombinant expression by
introducing codon changes and/or eliminating inhibitory regions in
the mRNA can be carried out by adapting the optimization methods
described in, e.g., U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664;
6,414,132; and 6,794,498, accordingly. For example, potential
splice sites and instability elements (e.g., A/T or A/U rich
elements) within the RNA can be mutated without altering the amino
acids encoded by the nucleic acid sequences to increase stability
of the RNA for recombinant expression. The alterations utilize the
degeneracy of the genetic code, e.g., using an alternative codon
for an identical amino acid. In some embodiments, it can be
desirable to alter one or more codons to encode a conservative
mutation, e.g., a similar amino acid with similar chemical
structure and properties and/or function as the original amino
acid. Such methods can increase expression of an anti-PSGL-1
tetravalent antibody or polypeptide thereof relative to the
expression of an anti-PSGL-1 tetravalent antibody or polypeptide
thereof encoded by polynucleotides that have not been optimized.
Furthermore, the polynucleotide sequences can be designed to match
the preferred codon usage in the host cell, e.g. E. coli codon
usage or CHO codon usage.
[0128] An optimized polynucleotide sequence encoding a tetravalent
antibody or polypeptide thereof described herein can hybridize to
an unoptimized polynucleotide sequence encoding a tetravalent
antibody or polypeptide thereof described herein. In specific
embodiments, an optimized nucleotide sequence encoding a
tetravalent antibody or polypeptide thereof described herein
hybridizes under high stringency conditions to an unoptimized
polynucleotide sequence encoding a tetravalent antibody or
polypeptide thereof described herein. In a specific embodiment, an
optimized nucleotide sequence encoding a tetravalent antibody or
polypeptide thereof described herein hybridizes under high
stringency, intermediate or lower stringency hybridization
conditions to an unoptimized nucleotide sequence encoding a
tetravalent antibody or polypeptide thereof described herein.
Information regarding hybridization conditions have been described,
see, e.g., U.S. Patent Application Publication No. US 2005/0048549
(e.g., paragraphs 72-73), which is incorporated herein by reference
in its entirety.
[0129] The polynucleotides of the present disclosure can be
obtained using chemical synthesis, recombinant methods, or PCR.
Methods of chemical polynucleotide synthesis are well known in the
art and need not be described in detail herein. One of skill in the
art can use the sequences provided herein and a commercial DNA
synthesizer to produce a desired DNA sequence.
[0130] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides can be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating, or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al. (1989).
[0131] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, as well as
PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer
Press, Boston (1994).
[0132] The present disclosure also provides vectors (e.g., cloning
vectors or expression vectors) comprising a nucleic acid sequence
encoding any of the polypeptides (including antibodies) described
herein. Suitable cloning vectors can be constructed according to
standard techniques or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Strategene, and
Invitrogen.
[0133] Expression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide according to the present
disclosure. The expression vector may replicable in the host cells
either as episomes or as an integral part of the chromosomal DNA.
Suitable expression vectors include but are not limited to
plasmids, viral vectors, including adenoviruses, adeno-associated
viruses, retroviruses, cosmids, and expression vector(s) disclosed
in PCT Publication No. WO 87/04462. Vector components may generally
include, but are not limited to, one or more of the following: a
signal sequence; an origin of replication; one or more marker
genes; and suitable transcriptional controlling elements (such as
promoters, enhancers, or terminator). For expression (i.e.,
translation), one or more translational controlling elements are
also usually required, such as ribosome binding sites, translation
initiation sites, or stop codons.
[0134] Methods of making antibodies and polypeptides derived from
the antibodies are known in the art and are disclosed herein.
Well-established methods may be used to identify anti-PSGL
antibodies (e.g., antibodies that specifically bind to human
PSGL-1), from which variable domains (e.g., VH and/or VL domains)
may be used in the tetravalent antibodies of the present
disclosure. Exemplary anti-human PSGL-1 antibodies, as well as
methods for screening, producing, and purifying such antibodies,
are described in International Application Pub. No. WO
2012/174001.
[0135] Additional anti-human PSGL-1 antibodies may be identified
using methods known in the art, such as those described in
International Application Pub. No. WO 2012/174001 and supra. For
example, the monoclonal antibodies can be prepared using hybridoma
technology, such as those described by Kohler and Milstein (1975),
Nature, 256:495. In a hybridoma method, a mouse, a hamster, or
other appropriate host animal, is typically immunized with an
immunizing agent (e.g., a cell expressing human PSGL-1 or a
fragment thereof) to elicit lymphocytes that produce or are capable
of producing antibodies that specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in vitro.
The lymphocytes are then fused with an immortalized cell line using
a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, Academic Press, (1986) pp. 59-1031). Immortalized cell
lines are usually transformed mammalian cells, particularly myeloma
cells of rodent, rabbit, bovine, or human origin. Usually, rat or
mouse myeloma cell lines are employed. The hybridoma cells may be
cultured in a suitable culture medium that desirably contains one
or more substances that inhibit the growth or survival of the
unfused, immortalized cells. For example, if the parental cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
includes hypoxanthine, aminopterin, and thymidine ("HAT medium"),
which substances prevent the growth of HGPRT-deficient cells.
[0136] Desired immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More desirable immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol. (1984), 133:3001; Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0137] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies. The
antibody may be screened for having specific binding to an ORP150
polypeptide (such as binding to an epitope in an extracellular
domain of the ORP150 polypeptide) obtained from or expressed on the
cell surface of plasmacytoma, multiple myeloma, colorectal,
gastric, or esophageal cancer or tumor cells. Cancer cells or an
ORP150 polypeptide (or a fragment thereof containing an
extracellular domain of an ORP150 polypeptide) may be used for
screening. For example, RPMI8226, U266, NCI-H929, L363, Colo205,
DLD-1, HT29, SNU-1, Kato-III, or CE146T cells may be used for
screening. A polypeptide comprising amino acids 673-800, 701-800,
673-752, or 723-732 of SEQ ID NO:17 may also be used for
screening.
[0138] In some embodiments, the binding specificity of monoclonal
antibodies produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (MA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard (1980),
Anal. Biochem., 107:220.
[0139] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium or
RPMI-1640 medium. Alternatively, the hybridoma cells may be grown
in vivo as ascites in a mammal.
[0140] The monoclonal antibodies can be generated by culturing the
hybridoma cells, and the antibodies secreted by the hybridoma cells
may further be isolated or purified. Antibodies may be isolated or
purified from the culture medium or ascites fluid by conventional
immunoglobulin purification procedures such as, for example,
protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0141] The tetravalent antibodies or polypeptides of the present
disclosure may be generated by screening a library of antibodies or
polypeptides to select antibodies or polypeptides that bind to
human PSGL-1, e.g., expressed on the cell surface of a cell.
Antibody phage display libraries known in the art may be used. In
some embodiments, the antibodies in the library (e.g., displayed on
phage) are single-chain Fv (scFv) fragments or Fab fragment. In
some embodiments, the antibodies in the library (e.g., displayed on
phage) are single-domain antibodies. For example, a single-domain
antibody may comprise all or a portion of the heavy chain variable
domain or all or a portion of the light chain variable domain of an
antibody. In some embodiments, the antibodies in the library are
human antibodies. The antibodies identified may further be tested
for their capabilities to induce cell death (e.g., apoptosis)
and/or bind human PSGL-1 using methods known in the art and
described herein.
[0142] The tetravalent antibodies of the present disclosure can be
made by recombinant DNA methods, such as those described in U.S.
Pat. Nos. 4,816,567 and 6,331,415. For example, DNA encoding the
variable or constant region of any of the tetravalent antibodies of
the present disclosure (or single, heavy, or light chain
polypeptides that are constituents thereof) can be readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells of the present disclosure serve as a preferred
source of such DNA. Once isolated, the DNA can be placed into
expression vectors, which are then transfected into host cells such
as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma
cells that do not otherwise produce immunoglobulin protein to
synthesize monoclonal antibodies in the recombinant host cells. The
DNA also can be modified, for example, by substituting the coding
sequence for human heavy and light chain constant domains in place
of the homologous murine sequences (U.S. Pat. No. 4,816,567) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the present disclosure, or can
be substituted for the variable domains of one antigen-combining
site of an antibody of the present disclosure to create a chimeric
bivalent antibody.
[0143] In some embodiments, the tetravalent antibodies of the
present disclosure are expressed from two expression vectors. For
example, each expression vector may express one monomer of a dimer
of the present disclosure (e.g., a single-chain polypeptide or
antibody heavy or light chain polypeptide). Alternatively, both
monomers of a dimer of the present disclosure are expressed from a
single expression vector.
[0144] Normally the expression vector has transcriptional and
translational regulatory sequences which are derived from a species
compatible with a host cell. In addition, the vector ordinarily
carries a specific gene(s) which is (are) capable of providing
phenotypic selection in transformed cells.
[0145] A wide variety of recombinant host-vector expression systems
for eukaryotic cells are known and can be used in the present
disclosure. For example, Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among eukaryotic
microorganisms, although a number of other strains, such as Pichia
pastoris, are available. Cell lines derived from multicellular
organisms such as Sp2/0 or Chinese Hamster Ovary (CHO), which are
available from the ATCC, may also be used as hosts. Typical vector
plasmids suitable for eukaryotic cell transformations are, for
example, pSV2neo and pSV2gpt (ATCC), pSVL and pSVK3 (Pharmacia),
and pBPV-1/pML2d (International Biotechnology, Inc.).
[0146] The eukaryotic host cells useful in the present disclosure
are, for example, hybridoma, myeloma, plasmacytoma, or lymphoma
cells. However, other eukaryotic host cells may be suitably
utilized provided the mammalian host cells are capable of
recognizing transcriptional and translational DNA sequences for
expression of the proteins; processing the leader peptide by
cleavage of the leader sequence and secretion of the proteins; and
providing post-translational modifications of the proteins, e.g.,
glycosylation.
[0147] Accordingly, the present disclosure provides host cells
(e.g., eukaryotic host cells) which are transformed by recombinant
expression vectors comprising DNA constructs disclosed herein and
which are capable of expressing the tetravalent antibodies or
polypeptides of the present disclosure. In some embodiments, the
transformed host cells of the present disclosure comprise at least
one DNA construct comprising a polynucleotide of the present
disclosure, or a polynucleotide expressing a monomer, dimer, or
tetravalent antibody of the present disclosure, and transcriptional
and translational regulatory sequences which are positioned in
relation to the coding DNA sequences to direct expression of
antibodies or polypeptides.
[0148] Any host cells capable of over-expressing heterologous DNAs
can be used for the purpose of isolating the genes encoding the
antibody, polypeptide, or protein of interest. Non-limiting
examples of mammalian host cells include but not limited to COS,
HeLa, and CHO cells. See also PCT Publication No. WO 87/04462.
Suitable non-mammalian host cells include prokaryotes (such as E.
coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe, or
K. lactis).
[0149] The host cells used in the present disclosure may be
transformed in a variety of ways by standard transfection
procedures well known in the art. Among the standard transfection
procedures which may be used are electroporation techniques,
protoplast fusion and calcium-phosphate precipitation techniques.
Such techniques are generally described by F. Toneguzzo et al.
(1986), Mol. Cell. Biol., 6:703-706; G. Chu et al., Nucleic Acid
Res. (1987), 15:1311-1325; D. Rice et al., Proc. Natl. Acad. Sci.
USA (1979), 79:7862-7865; and V. Oi et al., Proc. Natl. Acad. Sci.
USA (1983), 80:825-829. The vectors containing the polynucleotides
of interest can be introduced into the host cell by any of a number
of appropriate means, including electroporation, transfection
employing calcium chloride, rubidium chloride, calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; and infection (e.g., where the vector is an infectious
agent such as vaccinia virus). The choice of introducing vectors or
polynucleotides often depends on features of the host cell.
[0150] In the case of two expression vectors, the two expression
vectors can be transferred into a host cell one by one separately
or together (co-transfer or co-transfect).
[0151] The present disclosure also provides a method for producing
the antibodies or polypeptides that comprises culturing a host cell
comprising an expression vector(s) encoding the antibodies or the
polypeptides, and recovering the antibodies or polypeptides from
the culture by ways well known to one skilled in the art.
[0152] Furthermore, the desired antibodies can be produced in a
transgenic animal. A suitable transgenic animal can be obtained
according to standard methods which include micro-injecting into
eggs the appropriate expression vectors, transferring the eggs into
pseudo-pregnant females, and selecting a descendant expressing the
desired antibody.
[0153] The present disclosure also provides chimeric tetravalent
antibodies that specifically bind human PSGL-1. For example, the
variable and constant regions of the tetravalent antibody are from
separate species. In some embodiments, the variable regions of both
heavy chain and light chain are from the murine antibodies
described herein. The chimeric antibody of the present disclosure
can be prepared by techniques well-established in the art. See for
example, U.S. Pat. Nos. 6,808,901; 6,652,852; 6,329,508; 6,120,767;
and 5,677,427, each of which is hereby incorporated by reference.
In general, the chimeric antibody can be prepared by obtaining
cDNAs encoding the heavy and light chain variable regions of the
antibodies, inserting the cDNAs into an expression vector, which
upon being introduced into eukaryotic host cells, expresses the
chimeric antibody of the present disclosure. Desirably, the
expression vector carries a functionally complete constant heavy or
light chain sequence so that any variable heavy or light chain
sequence can be easily inserted into the expression vector.
[0154] The present disclosure provides a humanized tetravalent
antibody that specifically binds to human PSGL-1. The humanized
antibody is typically a human antibody in which residues from CDRs
are replaced with residues from CDRs of a non-human species such as
mouse, rat, or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
antibody are replaced by corresponding non-human residues.
[0155] There are four general steps to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains, (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process, (3) the actual humanizing
methodologies/techniques, and (4) the transfection and expression
of the humanized antibody. See, for example, U.S. Pat. Nos.
4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;
5,693,762; 5,585,089; 6,180,370; and 6,548,640. For example, the
constant region may be engineered to more resemble human constant
regions to avoid immune response if the antibody is used in
clinical trials and treatments in humans. See, for example, U.S.
Pat. Nos. 5,997,867 and 5,866,692.
[0156] It is important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, humanized antibodies can be
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three dimensional
models of the parental and humanized sequences. Three dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the consensus and
import sequence so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the CDR residues are directly and most substantially
involved in influencing antigen binding. The humanized antibodies
may also contain modifications in the hinge region to improve one
or more characteristics of the antibody.
[0157] In another alternative, antibodies may be screened and made
recombinantly by phage display technology. See, for example, U.S.
Pat. Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter
et al., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, the
phage display technology (McCafferty et al., Nature 348:552-553
(1990)) can be used to produce human antibodies and antibody
fragments in vitro, from immunoglobulin variable (V) domain gene
repertoires from unimmunized donors. According to this technique,
antibody V domain genes are cloned in-frame into either a major or
minor coat protein gene of a filamentous bacteriophage, such as M13
or fd, and displayed as functional antibody fragments on the
surface of the phage particle. Because the filamentous particle
contains a single-stranded DNA copy of the phage genome, selections
based on the functional properties of the antibody also result in
selection of the gene encoding the antibody exhibiting those
properties. Thus, the phage mimics some of the properties of the
B-cell. Phage display can be performed in a variety of formats; for
review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current
Opinion in Structural Biology 3, 564-571 (1993). Several sources of
V-gene segments can be used for phage display. Clackson et al.,
Nature 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed, and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Mark et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). In a natural immune response, antibody
genes accumulate mutations at a high rate (somatic hypermutation).
Some of the changes introduced will confer higher affinity, and
B-cells displaying high-affinity surface immunoglobulin are
preferentially replicated and differentiated during subsequent
antigen challenge. This natural process can be mimicked by
employing the technique known as "chain shuffling." Marks et al.,
Bio/Technol. 10:779-783 (1992)). In this method, the affinity of
"primary" human antibodies obtained by phage display can be
improved by sequentially replacing the heavy and light chain V
region genes with repertoires of naturally occurring variants
(repertoires) of V domain genes obtained from unimmunized donors.
This technique allows the production of antibodies and antibody
fragments with affinities in the pM-nM range. A strategy for making
very large phage antibody repertoires (also known as "the
mother-of-all libraries") has been described by Waterhouse et al.,
Nucl. Acids Res. 21:2265-2266 (1993). Gene shuffling can also be
used to derive human antibodies from rodent antibodies, where the
human antibody has similar affinities and specificities to the
starting rodent antibody. According to this method, which is also
referred to as "epitope imprinting," the heavy or light chain V
domain gene of rodent antibodies obtained by phage display
technique is replaced with a repertoire of human V domain genes,
creating rodent-human chimeras. Selection on antigen results in
isolation of human variable regions capable of restoring a
functional antigen-binding site, i.e., the epitope governs
(imprints) the choice of partner. When the process is repeated in
order to replace the remaining rodent V domain, a human antibody is
obtained (see PCT Publication No. WO 93/06213, published Apr. 1,
1993). Unlike traditional humanization of rodent antibodies by CDR
grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin. It is
apparent that although the above discussion pertains to humanized
antibodies, the general principles discussed are applicable to
customizing antibodies for use, for example, in dogs, cats,
primates, equines, and bovines.
[0158] In certain embodiments, the antibody is a fully human
antibody. Non-human antibodies that specifically bind an antigen
can be used to produce a fully human antibody that binds to that
antigen. For example, the skilled artisan can employ a chain
swapping technique, in which the heavy chain of a non-human
antibody is co-expressed with an expression library expressing
different human light chains. The resulting hybrid antibodies,
containing one human light chain and one non-human heavy chain, are
then screened for antigen binding. The light chains that
participate in antigen binding are then co-expressed with a library
of human antibody heavy chains. The resulting human antibodies are
screened once more for antigen binding. Techniques such as this one
are further described in U.S. Pat. No. 5,565,332. In addition, an
antigen can be used to inoculate an animal that is transgenic for
human immunoglobulin genes. See, e.g., U.S. Pat. No. 5,661,016.
[0159] The present disclosure also provides bispecific antibodies.
A bispecific antibody has binding specificities for at least two
different antigens (including different epitopes). In some
embodiments, a bispecific antibody of the present disclosure
includes two or more different VH and/or VL domains that
specifically bind PSGL-1. In some embodiments, the two or more
different VH and/or VL domains specifically bind the same epitope
of PSGL-1. In some embodiments, the two or more different VH and/or
VL domains specifically bind different epitopes of PSGL-1, which
may or may not be overlapping epitopes.
[0160] A bispecific antibody (a monoclonal antibody that has
binding specificities for at least two different antigens) can be
prepared using the antibodies disclosed herein. Methods for making
bispecific antibodies are known in the art (see, e.g., Suresh et
al., 1986, Methods in Enzymology 121:210). Traditionally, the
recombinant production of bispecific antibodies was based on the
coexpression of two immunoglobulin heavy chain-light chain pairs,
with the two heavy chains having different specificities (Millstein
and Cuello, 1983, Nature 305, 537-539). In some embodiments, a
bispecific tetravalent antibody may be produced using the methods
exemplified supra.
[0161] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. In some embodiments, the fusion is with
an immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2, and CH3 regions. In some embodiments, the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, is present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0162] Heteroconjugate antibodies, comprising two covalently joined
monomers or antibodies, are also within the scope of the present
disclosure. Such antibodies have been used to target immune system
cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (PCT Publication Nos. WO 91/00360 and WO
92/200373; and EP 03089). Heteroconjugate antibodies may be made
using any convenient cross-linking methods. Suitable cross-linking
agents and techniques are well known in the art, and are described
in U.S. Pat. No. 4,676,980.
[0163] Certain aspects of the present disclosure relate to antibody
variable domains and/or antibody fragments, e.g., that may be used
as a constituent of a tetravalent antibody described herein.
Antibody fragments may contain the active binding region of the
antibodies, such as Fab, F(ab').sub.2, scFv, Fv fragments, and the
like. Various methods known in the art may be used to produce
and/or isolate antibody fragments, which may be incorporated into a
tetravalent antibody of the present disclosure, e.g., by standard
recombinant techniques known in the art based on the concepts
described herein.
[0164] Single-chain Fv fragments may be produced, such as described
in Iliades et al., 1997, FEBS Letters, 409:437-441. Coupling of
such single-chain fragments using various linkers is described in
Kortt et al., 1997, Protein Engineering, 10:423-433. A variety of
techniques for the recombinant production and manipulation of
antibodies are well known in the art. Such fragments can be
produced from the monoclonal antibodies described herein using
techniques well established in the art (Rousseaux et al. (1986), in
Methods Enzymol., 121:663-69 Academic Press).
[0165] Methods of preparing antibody fragments are well known in
the art. For example, an antibody fragment can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 100 Kd
fragment denoted F(ab').sub.2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 50 Kd Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using papain produces two
monovalent Fab fragments and an Fc fragment directly. These methods
are described, for example, by U.S. Pat. Nos. 4,036,945 and
4,331,647 and references contained therein, which patents are
incorporated herein by reference. Also, see Nisonoff et al. (1960),
Arch Biochem. Biophys. 89: 230; Porter (1959), Biochem. J. 73: 119;
Smyth (1967), Methods in Enzymology 11: 421-426. Alternatively, the
Fab can be produced by inserting DNA encoding Fab of the antibody
into an expression vector for prokaryote or an expression vector
for eukaryote, and introducing the vector into a prokaryote or
eukaryote to express the Fab.
IV. Methods and Uses
[0166] Certain aspects of the present disclosure relate to methods
and uses for the tetravalent antibodies described herein. These
methods and uses are based at least in part on the properties of
the tetravalent antibodies as described herein, including without
limitation their increased number of epitope binding domains,
potential for lesser dependence upon cross-linking in vitro and/or
in vivo, differential potency for inducing apoptosis (e.g., of
human PSGL-1 expressing cells), and/or enhanced in vivo or trans
vivo efficacy.
[0167] As described herein, PSGL-1 is known to be involved in
inflammation and T cell biology. The tetravalent antibodies of the
present disclosure that specifically bind human PSGL-1 may find
use, inter alia, in treating individuals with diseases related to T
cell function (e.g., a T-cell mediated inflammatory disease), or
individuals in need of medical procedures that may result in
inflammatory conditions such as immunological reactions, or for
which such conditions are managed beforehand (e.g., a
transplantation or transfusion).
[0168] In some embodiments, a disorder or disease treated by the
methods described herein may be a T-cell mediated inflammatory
disease. Non-limiting examples of disorders and diseases that can
be treated, or one or more of whose symptoms may be ameliorated or
prevented using the tetravalent antibodies described herein
described herein include psoriasis, Crohn's disease, ankylosing
spondylitis, arthritis (including rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, and psoriatic arthritis),
diabetes mellitus, multiple sclerosis, encephalomyelitis,
myasthenia gravis, systemic lupus erythematosus, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), Sjogren's Syndrome, aphthous ulcer, iritis,
conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory
bowel diseases, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, graft versus host
disease (GVHD), sarcoidosis, primary biliary cirrhosis, uveitis
posterior, interstitial lung fibrosis, allergies such as atopic
allergy, AIDS, and T cell neoplasms such as leukemias or lymphomas.
In some embodiments, the disease is an autoimmune disease.
[0169] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is plaque psoriasis.
Plaque psoriasis or psoriasis vulgaris is the most common form of
psoriasis and is characterized by sharply demarcated, raised
erythematous skin plaques covered by silvery scale. There is a
predilection of the lesions to involve the extensor surfaces of the
extremities, the lumbosacral area, and the scalp. The corresponding
histopathological findings include significant inflammatory
cellular infiltration of the dermis and epidermis, increased
numbers of dilated vessels, and a substantial thickening of the
epidermis with disordered differentiation of keratinocytes and
hyperkeratosis. Approximately one third of patients with plaque
psoriasis are categorized as having moderate or severe disease and
are consequently candidates for therapy beyond just topical
treatment.
[0170] In another embodiment, the disorder treated in accordance
with the methods described herein is chronic plaque psoriasis.
Symptoms of plaque chronic psoriasis include, but are not limited
to, single or multiple raised reddened patches of skin, ranging
from coin-sized to larger, on any part of the body, including but
not limited to the knees, elbows, lumbosacral regions, scalp, and
nails.
[0171] In another embodiment, the disorder treated in accordance
with the methods described herein is guttate psoriasis. Symptoms of
guttate psoriasis include, but are not limited to, flares of water
drop shaped scaly plaques on the skin, followed by an infection,
such as a streptococcal throat infection.
[0172] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is inverse psoriasis.
Symptoms of inverse psoriasis include, but are not limited to,
smooth, usually moist areas of skin that are red and inflamed,
unlike the scaling associated with plaque psoriasis, on one or more
of the following body parts: armpits, groin, under the breasts, and
in other skin folds around the genitals and buttocks.
[0173] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is pustular psoriasis.
Symptoms of pustular psoriasis include, but are not limited to,
pus-filled blisters that vary in size and location, but mostly on
the hands and feet.
[0174] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is erythodermic
psoriasis. Symptoms of erythodermic psoriasis include, but are not
limited to, periodic, widespread, fiery redness of the skin and the
shedding of scales in sheets, rather than smaller flakes. The
reddening and shedding of the skin are often accompanied by severe
itching and pain, heart rate increase, and fluctuating body
temperature.
[0175] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is rheumatoid
arthritis. Symptoms of rheumatoid arthritis, include, but are not
limited to, fatigue, loss of appetite, low fever, swollen glands,
weakness, joint pain in wrists, elbows, shoulders, hips, knees,
ankles, toes, jaw, hands, feet, fingers, and/or neck, morning
stiffness, chest pain when taking a breath (pleurisy), eye burning,
itching, and discharge, nodules under the skin, numbness, tingling,
or burning in the hands and feet.
[0176] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is Crohn's disease.
Symptoms of Crohn's disease, but are not limited to, crampy
abdominal (belly area) pain, fever, fatigue, loss of appetite, pain
with passing stool (tenesmus), persistent, watery diarrhea,
unintentional weight loss, constipation, eye inflammation, fistulas
(usually around the rectal area, may cause draining of pus, mucus,
or stools), joint pain, liver inflammation, mouth ulcers, rectal
bleeding and bloody stools, skin lumps or sores (ulcers), and
swollen gums.
[0177] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is ankylosing
spondylitis. Symptoms of ankylosing spondylitis include, but are
not limited to, frequent pain and stiffness in the lower back and
buttocks, spine, and/or neck; and pain and tenderness spreading to
the ribs, shoulder blades, hips, thighs and heels; inflammation of
the eye (iridocyclitis and uveitis), causing redness, eye pain,
vision loss, floaters and photophobia; fatigue; and nausea.
[0178] In another embodiment, the disease or disorder treated in
accordance with the methods described herein is diabetes mellitus.
Symptoms of diabetes mellitus include, but are not limited to, loss
of weight, polyuria (frequent urination), polydipsia (increased
thirst), polyphagia (increased hunger), cardiovascular disease,
diabetic retinopathy, diabetic neuropathy, hyperosmolar nonketotic
state, and diabetic ketoacidosis.
[0179] In some embodiments, a tetravalent antibody or composition
of the present disclosure may be administered to the individual
before, concurrently with, and/or after a transplantation. For
example, as described in greater detail below, a tetravalent
antibody or composition of the present disclosure may be
administered to increase the likelihood of a favorable treatment
outcome, decrease the likelihood of an unfavorable outcome, and/or
mitigate or prevent symptoms or unfavorable outcomes occurring
before, concurrently with, or after the transplantation has been
completed.
[0180] As used herein, treating an individual in need of a
transplantation may refer to one or more of therapeutic treatment
and prophylactic or preventative measures (e.g., increasing the
likelihood of a favorable treatment outcome, such as graft
survival, graft function, or decreasing the likelihood of an
unfavorable outcome, such as an unfavorable response to treatment,
or a condition that reduces the likelihood a favorable treatment,
such as a transplantation, from occurring). Treating may include
without limitation mitigating or preventing conditions and symptoms
associated with a disorder or a condition, and/or problems or
conditions that interfere with or limit an individual's access to
treatment options of a disorder or a condition, such as
sensitization, hypersensitization, high panel reactive antibodies
(PRA) level and/or presence of pre-existing alloantibodies that
limit availability of grafts to an individual awaiting a
transplantation. Those in need of treatment include those already
with the disorder or condition, as well as those in which the
disorder or condition is to be prevented. Treatment of a disorder
or condition may suppress immune-mediated events associated with
the disorder or condition, ameliorate the symptoms of the disorder
or condition, reduce the severity of the disorder or condition,
alter the course of the disorder or condition progression, and/or
ameliorate or cure the basic disorder or condition.
[0181] For example, successful treatment of an individual awaiting
transplantation include, but is not limited to, reducing the level
of alloantibodies, reducing panel reactive antibodies (PRA),
enabling the individual to have more cross-match compatible donors,
increasing the likelihood or probability of the individual to
receive a graft, shortening the expected waiting period of the
individual for a graft, desensitizing the individual, lowering risk
of transplant-associated symptoms or conditions (such as
immune-mediated events as described below), or any combination
thereof.
[0182] For example, successful treatment of an individual receiving
a transplantation includes, but is not limited to, protection and
maintenance of the transplanted organ or tissue for a long term,
which comprises controlling, reversing, mitigating, delaying, or
preventing one or more symptoms or undesirable conditions
associated with the organ transplant, such as immune-mediated
events, including, but not limited to, production of donor-specific
alloantibodies (DSA), GVHD, antibody-mediated rejection (AMR),
hyperacute graft rejection, chronic graft rejection, graft failure,
and graft loss, as measured by functional or histological signs of
the symptom or condition. A treatment capable of controlling a
disorder or condition (e.g., graft rejection) may include a
treatment that slows the progression of the disease process, when
initiated after functional or histological signs of the disorder or
condition (e.g., graft rejection) are observed. Further, a
treatment capable of reversing a disease or condition (e.g., graft
rejection) may include a treatment that, when initiated after
functional or histological signs of the disease or condition (e.g.,
graft rejection) have appeared, reverses the disease process and
returns functional and histological findings closer to normal. A
treatment capable of "delaying progression" of a disorder or
condition (e.g., graft rejection) may include deferring, hindering,
slowing, retarding, stabilizing, and/or postponing development of
the disorder or condition (e.g., graft rejection). This delay can
be of varying lengths of time, depending on the history of the
disease and/or individual being treated. As is evident to one
skilled in the art, a sufficient or significant delay can, in
effect, encompass prevention, in that the individual, e.g., an
individual at risk for developing the disorder or condition, does
not develop the disorder or condition.
[0183] In some embodiments, a transplantation of the present
disclosure may be transplantation of one or more tissues or organs
including without limitation bone marrow, kidney, heart, liver,
neuronal tissue, lung, pancreas, skin, and intestine (e.g., small
and/or large intestine, as well as any sub-tissues thereof).
[0184] In addition, tetravalent antibodies are useful for
preventing and/or treating certain disorders and diseases
associated with or caused (in whole or in part) by increased
proliferation and/or numbers of activated T cells relative to the
proliferation and/or numbers of activated T cells found in healthy
individuals or individuals not having the particular disorder or
disease. Non-limiting examples of disorders and diseases that can
be prevented and/or treated using the tetravalent antibodies
described herein include graft-versus-host disease and cases of
transplantation rejection (including transplantation rejection
using allogeneic or xenogeneic tissues) such as bone marrow
transplantation, liver transplantation, kidney transplant, or the
transplantation of any organ or tissue.
[0185] In some embodiments, a tetravalent antibody or composition
of the present disclosure may be administered to the individual
before, concurrently with, and/or after a transfusion. For example,
as described in greater detail below, a tetravalent antibody or
composition of the present disclosure may be administered to
increase the likelihood of a favorable treatment outcome, decrease
the likelihood of an unfavorable outcome, and/or mitigate or
prevent symptoms occurring before, concurrently with, or after the
transfusion has been completed.
[0186] As used herein, treating an individual in need of a
transfusion may refer to one or more of therapeutic treatment and
prophylactic or preventative measures (e.g., increasing the
likelihood of a favorable treatment outcome, such as replacement or
supplementation of blood components/cells, or decreasing the
likelihood of an unfavorable outcome, such as an unfavorable
response to treatment, inefficacy of treatment, or immunological
reaction, or a condition that reduces the likelihood a favorable
treatment, such as a transfusion, from occurring). Treating may
include without limitation mitigating or preventing conditions and
symptoms associated with a disorder or a condition, and/or problems
or conditions that interfere with or limit an individual's access
to treatment options of a disorder or a condition. Those in need of
treatment include those already with the disorder or condition, as
well as those in which the disorder or condition is to be
prevented. Treatment of a disorder or condition may suppress
immune-mediated events associated with the disorder or condition,
ameliorate the symptoms of the disorder or condition, reduce the
severity of the disorder or condition, alter the course of the
disorder or condition progression, and/or ameliorate or cure the
basic disorder or condition.
[0187] In some embodiments, the transfusion is a transfusion
comprising one or more of white blood cells, red blood cells, and
platelets. In some embodiments, the transfusion comprises whole
blood or one or more blood products, including without limitation
white blood cells, red blood cells, platelets, fresh frozen plasma,
cryoprecipitate or blood clotting factors, antibodies, and/or blood
substitutes. Exemplary conditions that may be treated with a
transfusion (e.g., transfusion of blood or a blood product) include
without limitation hemorrhage or blood loss, reduced hematocrit or
hemoglobin (e.g., anemia), sickle cell disease, thalassemia, blood
supplementation during or after surgical procedures, cardiac
disease, traumatic injury, deficiency of one or more blood factors
(e.g., hemophilia, von Willebrand disease, hypofibrinogenemia, or a
deficiency in factor II, V, VII, IX, X, or XI), conditions
requiring fibrinogen supplementation (e.g., liver disease, blood
transfusion, etc.), bone marrow failure, platelet function
disorders, thrombocytopenia, immunodeficiency (e.g., from a therapy
or disease), and the like. Descriptions of practices, dosing,
responses, indications, and preparations related to transfusions
may be found, e.g., in the American Red Cross Compendium of
Transfusion Practice Guidelines.
[0188] Administration of a tetravalent antibody or polypeptide in
accordance with the methods described herein can be continuous or
intermittent, depending, for example, upon the recipient's
physiological condition, whether the purpose of the administration
is therapeutic or prophylactic, and other factors known to skilled
practitioners. The administration of an antibody or a polypeptide
may be essentially continuous over a preselected period of time or
may be in a series of spaced dose.
[0189] The dosage and frequency of administration of a tetravalent
antibody described herein or a pharmaceutical composition thereof
is administered in accordance with the methods for preventing
and/or treating while minimizing side effects. The exact dosage of
a tetravalent antibody described herein to be administered to a
particular subject or a pharmaceutical composition thereof can be
determined by a practitioner, in light of factors related to the
subject that requires treatment. Factors which can be taken into
account include the severity of the disease state, general health
of the subject, age, and weight of the subject, diet, time and
frequency of administration, combination(s) with other therapeutic
agents or drugs, reaction sensitivities, and tolerance/response to
therapy. The dosage and frequency of administration of a
tetravalent antibody described herein or a pharmaceutical
composition thereof can be adjusted over time to provide sufficient
levels of the antibody or an antibody derived antigen-binding
fragment, or to maintain the desired effect.
[0190] The precise dose to be employed in the formulation will also
depend on the route of administration, and the seriousness of an
inflammatory disorder or disease, and should be decided according
to the judgment of the practitioner and each patient's
circumstances.
[0191] Effective doses can be extrapolated from dose-response
curves derived from in vitro or animal model test systems.
[0192] In one embodiment, any of the compositions described herein
is formulated for administration by intraperitoneal, intravenous,
subcutaneous, or intramuscular injections, or other forms of
administration such as oral, mucosal, via inhalation, sublingually,
etc. Parenteral administration, in one embodiment, is characterized
by injection, either subcutaneously, intramuscularly or
intravenously is also contemplated herein. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. The injectables,
solutions and emulsions also contain one or more excipients.
Suitable excipients are, for example, water, saline, dextrose,
glycerol or ethanol. In addition, if desired, the pharmaceutical
compositions to be administered can also contain minor amounts of
non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, stabilizers, solubility enhancers, and
other such agents. Other routes of administration may include,
enteric administration, intracerebral administration, nasal
administration, intraarterial administration, intracardiac
administration, intraosseous infusion, intrathecal administration,
intravenous infusion, subcutaneous implantation or injection,
intramuscular administration, intrarectal administration
intravaginal administration, intragastrical administration,
intratracheal administration, intrapulmonary administration and
intraperitoneal administration. Preparations for parenteral
administration include sterile solutions ready for injection,
sterile dry soluble products, such as lyophilized powders, ready to
be combined with a solvent just prior to use, including sterile
suspensions ready for injection, sterile dry insoluble products
ready to be combined with a vehicle just prior to use and sterile
emulsions. The solutions can be either aqueous or nonaqueous. If
administered intravenously, suitable carriers include physiological
saline or phosphate buffered saline (PBS), water, and solutions
containing thickening and solubilizing agents, such as glucose,
polyethylene glycol, and polypropylene glycol and mixtures
thereof.
[0193] In another embodiment, the present disclosure also
contemplates administration of a composition comprising the
antibodies or polypeptides of the present disclosure conjugated to
other molecules, such as detectable labels, or therapeutic or
cytotoxic agents. The agents may include, but are not limited to
radioisotopes, toxins, toxoids, inflammatory agents, enzymes,
antisense molecules, peptides, cytokines, and chemotherapeutic
agents. Methods of conjugating the antibodies with such molecules
are generally known to those of skilled in the art. See, e.g., PCT
publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.
5,314,995; and EP 396,387.
[0194] In one embodiment, the composition comprises an antibody or
polypeptide conjugated to a cytotoxic agent. Cytotoxic agents can
include any agents that are detrimental to cells. An exemplary
class of cytotoxic agents that can be conjugated to the antibodies
or fragments may include, but are not limited to, paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, teniposide, vincristine, vinblastine, colchicine,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, puromycin, and
analogs or homologs thereof.
V. Pharmaceutical Compositions
[0195] The present disclosure also provides pharmaceutical
compositions comprising tetravalent antibodies or polypeptides
described herein, and a pharmaceutically acceptable carrier or
excipients. The pharmaceutical compositions may find use, e.g., in
the methods, uses, and/or kits of the present disclosure.
[0196] Pharmaceutically acceptable carriers or excipients are known
in the art, and are relatively inert substances that facilitate
administration of a pharmacologically effective substance. For
example, an excipient can give form or consistency, or act as a
diluent. Suitable excipients include but are not limited to
stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers. In certain embodiments, a tetravalent
antibody described herein is in a liquid pharmaceutical
composition. Liquid pharmaceutically administrable compositions
can, for example, be prepared by dissolving, dispersing, or
otherwise mixing an antibody described herein in a carrier, such
as, for example, water, saline, aqueous dextrose, glycerol,
glycols, ethanol, and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be
administered can also contain minor amounts of nontoxic auxiliary
substances such as wetting agents, emulsifying agents, solubilizing
agents, and pH buffering agents and the like. Excipients as well as
formulations for parenteral and nonparenteral drug delivery are set
forth in Remington, The Science and Practice of Pharmacy 20th Ed.
Mack Publishing (2000).
[0197] The pharmaceutical compositions are provided for
administration to humans and animals in unit dosage forms, such as
sterile parenteral solutions or suspensions containing suitable
quantities of a tetravalent antibody described herein. The
tetravalent antibody is, in one embodiment, formulated and
administered in unit-dosage forms or multiple-dosage forms.
Unit-dose forms as used herein refers to physically discrete units
suitable for human and animal subjects and packaged individually as
is known in the art. Each unit-dose contains a predetermined
quantity of the antibody or the antibody derived antigen-binding
fragment sufficient to produce the desired therapeutic effect, in
association with the required pharmaceutical carrier, vehicle or
diluent. Examples of unit-dose forms include ampoules and syringes.
Unit-dose forms can be administered in fractions or multiples
thereof. A multiple-dose form is a plurality of identical
unit-dosage forms packaged in a single container to be administered
in segregated unit-dose form. Examples of multiple-dose forms
include vials, or bottles of pints or gallons. Hence, multiple dose
form is a multiple of unit-doses which are not segregated in
packaging.
[0198] The concentration of tetravalent antibody in the
pharmaceutical composition will depend on, e.g., the
physicochemical characteristics of the antibody or the antibody
derived antigen-binding fragment, the dosage schedule, and amount
administered as well as other factors known to those of skill in
the art. In some embodiments, the pharmaceutical compositions
provide a dosage of from about 0.001 mg to about 100 mg of
tetravalent antibody per kilogram of body weight per day.
Pharmaceutical dosage unit forms can be prepared to provide from
about 0.001 mg to about 100 mg, and/or a combination of other
optional essential ingredients per dosage unit form.
[0199] In some embodiments, the present disclosure provides
tetravalent antibodies and compositions (such as the pharmaceutical
compositions described herein) for use in any of the methods
described herein, whether in the context of use as a medicament
and/or use for manufacture of a medicament.
VI. Kits
[0200] Certain aspects of the present disclosure are related to
kits or articles of manufacture that comprise a tetravalent
antibody of the present disclosure. Optionally, the kits described
herein may contain one or more pharmaceutically acceptable
carriers, such as the exemplary carriers described herein. In some
embodiments, a kit of the present disclosure includes a
pharmaceutical composition of the present disclosure. Kits
described herein may find use, e.g., in the methods or uses of the
present disclosure.
[0201] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container. The containers may be unit doses, bulk packages
(e.g., multi-dose packages) or sub-unit doses. Instructions
supplied in the kits of the present disclosure are typically
written instructions on a label or package insert (e.g., a paper
sheet included in the kit), but machine-readable instructions
(e.g., instructions carried on a magnetic or optical storage disk)
are also acceptable.
[0202] In some embodiments, the kits further include a package
insert comprising instructions for administration of the
tetravalent antibody to treat a T-cell mediated inflammatory
disease. In some embodiments, the kits further include a package
insert comprising instructions for administration of the
tetravalent antibody before, concurrently with, and/or after a
transfusion or transplantation.
[0203] The kits of the present disclosure are in suitable
packaging. Suitable packaging includes, but is not limited to,
vials, bottles, jars, flexible packaging (e.g., sealed Mylar or
plastic bags), and the like. Also contemplated are packages for use
in combination with a specific device, such as an inhaler, nasal
administration device (e.g., an atomizer), or an infusion device
such as a minipump. A kit may have a sterile access port (for
example the container may be an intravenous solution bag or a vial
having a stopper pierceable by a hypodermic injection needle). The
container may also have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is a tetravalent antibody or
polypeptide described herein. The container may further comprise a
second pharmaceutically active agent. In some embodiments, a kit
may further include any other material or device useful in a
treatment (e.g., a transfusion or transplantation), including
without limitation one or more containers, tubing, sterilizing
agents or equipment, cannulae, syringes, and the like.
EXAMPLES
[0204] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1: Generation and Characterization of Anti-PSGL-1
Tetravalent Antibodies
[0205] P-selectin Glycoprotein Ligand-1 (PSGL-1) is expressed on a
wide range of hematopoietic cells, including myeloid, lymphoid,
dendritic, and CD34+ stem cell populations (see, e.g., Spertini et
al. 1996, J Cell Biol. 135(2):523-31). Several mouse antibodies
specific for PSGL-1 and capable of inducing apoptosis in T cells
have previously been identified. Among these mouse antibodies, an
antibody (h15A7) that did not interfere with the interaction
between P-selectin and PSGL-1, which required for efficient
localization of T cells and neutrophils to target inflammatory
tissues, was chosen for clinical development and was modified to a
humanized kappa-light-chain containing IgG4 monoclonal antibody to
minimize ADCC and CDC on PSGL-1 expressing cells (see, e.g., U.S.
Pat. No. 7,604,800). Subsequently, h15A7 was further engineered to
produce h15A7H, which has a mutation of SER228PRO in hinge region
of h15A7 (International Application Pub. No. WO 2012/174001). This
mutation was introduced in order to reduce antibody shuffling, the
intermolecular exchange among IgG4 antibodies in vivo. In vitro
studies showed that h15A7/h15A7H preferentially induced apoptosis
of late-stage activated T cells but not other PSGL-1-expressing
cells. Without wishing to be bound to theory, it is thought that
the mechanism of action of h15A7H appears to be dependent at least
in part on cross-linking of human PSGL-1 molecules, which is
mediated by antibody cross-linker in vitro and possibly
FcR-expressing cells in vivo.
[0206] The Example presented below describes the development of
several cross-linker/FcR-expressing cell-independent tetravalent
antibodies derived from h15A7H (FIGS. 1A & 1B). Without wishing
to be bound to theory, tetravalent antibodies may possess
advantages over h15A7H for clinical development, e.g., treatment of
T-cell mediated inflammatory diseases. These results demonstrate
that tetravalent h15A7H antibodies show enhanced efficacy compared
to the parental h15A7H antibody both in vitro and trans vivo.
[0207] Methods
Cells and Reagents
[0208] Sp2/0-Ag14 (ATCC.RTM.CRL-1581.TM.) and Sp2/0-hPSGL-1 were
cultured in 90% DMEM (GIBC.RTM., Cat. No. 11965-092.TM.)
supplemented with 10% FBS (GIBC.RTM., Cat. No. 26140-079), 100 U/mL
penicillin/100 .mu.g/mL streptomycin (GIBCO.RTM., Cat. No. 15140)
and 1 mM sodium pyruvate (GIBCO.RTM., Cat. No. 11360).
[0209] The h15A7H antibody was described in International
Application Pub. No. WO 2012/174001. The h15A7H tetravalent
antibodies used in the study were produced from a Flp-In CHO stable
cell line, purified by protein A affinity chromatography, and
maintained in Dulbecco's Phosphate-Buffered Saline (GIBCO.RTM. Cat.
No. 21600-069)/0.02% Tween-20 (JT Baker.RTM. X251-07). Human
IgG4p/K as irrelevant isotype control antibody was produced from
Flp-In CHO cells. 12H5.5 is a murine IgG1 anti-idiotype antibody
against h15A7/h15A7H.
Animals
[0210] Female B6 mice at 6-8 weeks of age were obtained from
BioLASCO Taiwan Co., Ltd, Taipei, Taiwan. All mice were maintained
under specific pathogen-free conditions. All animal studies were
conducted following the guidelines of the Institutional Animal Care
and Use Committee.
Construction of Anti-PSGL-1 Tetravalent Antibody Variants
[0211] scDb.sub.2-Fc
[0212] scDb.sub.2-Fc (FIG. 1A, left) included 2 domains of
single-chain diabodies (scDbs) fused in parallel to the N-terminals
of human IgG4 Fc with a mutation in the hinge region to minimize
half-antibody exchange in vivo. Each scDb domain contained not only
a domain sequence of VL-VH-VL-VH, but also a linker
(G.sub.4S.sub.1).sub.5 (SEQ ID NO:33) between VH and VL and two
identical linkers (e.g., SEQ ID NO:34) between VL and VH. Several
scDb-Fcs with said linkers of different length were generated for
optimization
[0213] taFv.sub.2-Fc
[0214] taFv.sub.2-Fc (FIG. 1A, middle) included 2 tandem
single-chain variable fragment (scFv) units (termed taFv for tandem
scFv) fused in parallel to the N-terminals of human IgG4 Fc with a
mutation in the hinge region to minimize half-antibody exchange in
vivo. There were three different kinds of scFvs used to construct
taFv, including v2 (VH-VL), v3 (VL-VH), and v4 (VL-VH) versions,
containing a linker (G.sub.4S.sub.1).sub.5 (SEQ ID NO:33) between
VH and VL. Among them, v2 and v4 were structure-constrained by the
formation of VH44-VL100 disulfide bond. The VH44-VL100 disulfide
bond was introduced into scFv in both VL-VH and VH-VL orientations
for increased conformational stability (see SEQ ID NOs:29 and 30).
Each taFv had either sequential v2-v3 or sequential v4-v2 of
anti-PSGL-1 scFv with a linker ASTGS (SEQ ID NO:27) between the two
scFvs.
[0215] scFv-IgG
[0216] The disulfide-constrained v2 version of anti-PSGL-1 scFv was
used to generate 3 scFv-IgG4p variants (FIG. 1A, right), including
scFv.sub.4-crIgG4p, scFv.sub.2-LC-IgG4p, and LC-scFv.sub.2-IgG4p.
scFv.sub.4-crIgG4p had 4 scFv units fused in parallel to the
N-terminals of both constant regions of kappa light chain and heavy
chain of IgG4p (crIgG) without a linker. scFv.sub.2-LC-IgG4p had 2
scFv units fused in parallel to the N-terminals of kappa light
chains of h15A7H IgG with a linker ASTGSG.sub.4S (SEQ ID NO:28)
in-between, whereas LC-scFv.sub.2-IgG4p had 2 scFv units fused in
parallel to the C-terminals of kappa light chains of h15A7H IgG
with a linker (G.sub.4S).sub.2 (SEQ ID NO:34) in-between. Light
chains of LC-scFv.sub.2 IgG4p and scFv.sub.2-LC IgG4p formats were
separately sub-cloned into a pcDNA5/FRT vector that encoded an
intact h15A7H heavy chain sequence for antibody expression. FIG. 1B
shows another diagram of these tetravalent antibody formats with
the variable fragments shaded.
[0217] cDNAs of all tetravalent antibodies were cloned into the
pcDNA5/FRT vector (Invitrogen.TM., Cat. No: V6010-20) for
tetravalent antibody expression.
Production of Stable Cell Lines Expressing Anti-PSGL-1 Tetravalent
Antibody Variants
[0218] Anti-PSGL1 tetravalent antibody variants were stably
expressed and produced in Flp-In CHO cells (Invitrogen.TM., Cat.
No: R708-07). The cDNA sequences of tetravalent antibody variants
were inserted into the pcDNA5/FRT vector (Invitrogen.TM., Cat. No:
V6010-20) and co-transfected with pOG44 (Invitrogen.TM., Cat. No
V6005-20) following the standard procedure provided by the vendor.
The culture supernatants of the established cell lines were
collected and purified with protein A sepharose beads (GE
Healthcare.TM., Cat. No: 17-5280-04). The purified proteins were
analyzed with both SDS-PAGE and size exclusion chromatography to
ensure the quality of antibodies.
Reducing and Non-Reducing SDS-PAGE (Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis)
[0219] Purified anti-PSGL-1 tetravalent antibodies were
electrophoresed in 10% reducing and non-reducing SDS polyacrylamide
gels. For the reducing SDS polyacrylamide gels, 2 .mu.g of antibody
were mixed with 5.times.SDS sample buffer (300 nM Tris, pH6.8, 10%
SDS, 50% glycerol, 5% 2-mercaptoethanol and 0.06% bromophenol blue)
and boiled for 10 min at 100.degree. C. before loading. For the
non-reducing SDS polyacrylamide gels, 2 .mu.g of antibodies were
mixed with 5.times. non-reducing sample buffer (300 nM Tris, pH6.8,
10% SDS, 50% glycerol and 0.06% bromophenol blue) and boiled for 10
min at 100.degree. C. before loading. The reducing and non-reducing
protein samples were loaded onto the same SDS-polyacrylamide gels
where electrophoresis was performed. Coomassie blue staining was
used to detect proteins on the gel after electrophoresis.
Binding Assay of Anti-PSGL-1 Tetravalent Antibody Variants
[0220] Sp2/0 cells transfected with human PSGL-1(Sp2/0-hPSGL1) were
used as the PSGL-1 expressing cell line. Sp2/0-hPSGL1 cells were
centrifuged at 1200 rpm for 5 min. The cell pellets were
resuspended in FACS buffer (PBS containing 1% FBS) and pipetted
into 96 well plate (1.times.10.sup.5 cells/well). To each well was
added 100 .mu.l of supernatants containing humanized
15A7H(h15A7H)/tetravalent antibodies, and these were incubated for
60 min at 4.degree. C. The cells were washed three times with cold
FACS buffer and then incubated with 100 .mu.l of Mouse Anti-Human
IgG.sub.4 pFc'-PE (SouthernBiotech Cat. no. 9190-09) at 1 .mu.g/ml
concentration for 60 min at 4.degree. C. Subsequently, the cells
were washed three times with cold FACS buffer and analyzed by FACS
analysis. All flow cytometric analyses were performed on a BD-LSR
flow cytometer (Becton Dickinson) using the Cell Quest
software.
Apoptosis Assay of Anti-PSGL-1 Tetravalent Antibody Variants
[0221] 1.times.10.sup.5 Sp2/0-hPSGL1 cells were seeded into the
wells of 96-well plates. Aliquots of purified anti-PSGL-1
tetravalent and control antibodies at titrated concentrations were
prepared freshly and added to each well. The treated cells were
kept at 37.degree. C. for 6 hr before FACS analysis for cellular
apoptosis assay.
[0222] For the cellular apoptosis assay, an Annexin-V-FITC
Apoptosis Detection Kit (Strong Biotech, Cat. No. AVK250) was used
following the manufacturer's instructions. In brief, the treated
cells were harvested and resuspended in 100 .mu.l Annexin V binding
buffer containing 0.5 .mu.l Annexin V-FITC at room temperature.
After 15 min incubation in the dark, the cells were washed twice
with 200 .mu.l of Annexin V binding buffer. Before FACS analysis, 1
.mu.l of propidium iodide (PI) per sample was added. All flow
cytometric analyses were performed on a BD-LSR flow cytometer
(Becton Dickinson) using Cell Quest software. The Annexin V
positive and/or PI positive cells are considered apoptotic
cells.
Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
[0223] 500 ml whole blood was collected from healthy donors that
were previously tested as good tetanus responders. The blood was
centrifuged at 1500 rpm for 6 min. The upper plasma layer was
discarded, and the remnant blood was diluted with an equivalent
volume of PBS. The diluted whole blood was carefully added over a
Ficoll (GE, Ficoll Plaque Plus, Cat #17-1440-02) layer and
centrifuged at 2400 rpm for 15 mins at room temperature. The buffy
coat layer containing mononuclear cells was collected and washed
with PBS 3 times to minimize platelet contamination. The cells were
resuspended in PBS and kept on ice before use.
Trans-Vivo Delayed Type Hypersensitivity (DTH)
[0224] 8-10.times.10.sup.6 PBMC cells, along with 0.25LF unit of
PBS-dialyzed Tetanus Toxoid (TT, Kuo Kwang, Cat # K4103-11) or PBS,
were injected in a final volume of 50 .mu.l into the hind footpad
of female B6 mice. Mice of 6.about.8 weeks were used in all
experiments. Footpad thickness was measured before and 24 hrs post
injection using a dial thickness gauge. The pre-injected value was
subtracted from post-injection value to obtain the net paw
thickness. All measurement values were recorded in millimeters
(mm). h15A7H and h15A7H tetravalent antibodies titrated in PBS were
intravenously administered at indicated doses into B6 mice one hour
prior to PBMC and TT injection. PBS was used as the vehicle
control. 2 or 4 paws (1 or 2 mice) per treatment were tested. The
plasma samples were collected 24 hrs after Ab administration to
check the concentrations of antibody variants. The percent
inhibition of paw thickness was calculated as follows:
100.times.(.DELTA. paw thicknesss.sub.veh-.DELTA. paw
thickness.sub.Ab)/(.DELTA. paw thickness.sub.veh-.DELTA. paw
thickness.sub.PBMC only).
ELISA for Detecting Antibody Concentration in Mouse Plasma
[0225] 96-well microtiter plates were coated with anti-idiotype
antibody 12H5.5 at 0.5 .mu.g/mL in ELISA coating buffer (30 mM
Na.sub.2CO.sub.3/100 mM NaHCO.sub.3) at 4.degree. C. overnight.
Plates were then blocked with 200 .mu.L/well of 0.5% BSA in PBS for
1 hour at room temperature, and washed 3 times with ELISA washing
buffer (0.05% Tween20 in PBS), followed by addition of 50
.mu.L/well of calibration standard or samples. Calibration
standards at a serial dilution were first prepared in the normal
mouse plasma. Calibration standard or samples were pre-diluted
1000.times. in assay diluent (0.1% BSA and 0.05% Tween 20 in PBS),
to make a final concentration of 0.1% mouse plasma in assay
diluents, before dispensing onto the plates. Subsequent dilutions,
if needed, were prepared using assay diluents containing 0.1%
normal mouse plasma. After 1 hour incubation at room temperature
and washing 5 times with ELISA washing buffer, the secondary
antibody mouse anti-human IgG.sub.4 pFc'-HRP (SouthernBiotech Cat.
no. 9190-05; dilution 1:15000) was added at 50 .mu.L/well and
incubated at room temperature for 1 hour. The plates were then
washed 5 times with ELISA washing buffer, followed by addition of
TMB substrate for color development. Reactions were stopped by 0.5N
H.sub.2SO.sub.4, and an absorbance value was measured at 450 nm in
a microtiter plate reader (Molecular Device VERSAmax).
[0226] Results
Reducing and Non-Reducing SDS-PAGE of Humanized 15A711 Tetravalent
Antibodies
[0227] As shown in FIGS. 2A-2C, SDS-PAGE followed by Coomassie blue
staining was used to verify the molecular weight and basic
structure of anti-PSGL-1 tetravalent antibodies under non-reducing
and reducing conditions. h15A7H V2-V3, V4-V2 and LH10-g4pFc under
non-reducing conditions yielded a major protein band with a
molecular weight of around 150 kDa (FIG. 2A). In the same
conditions, h15A7H scFv.sub.2-LC IgG4p, LC-scFv.sub.2IgG4p, and
scFv.sub.4-crIgG4p yielded a major protein band with a molecular
weight of around 200 kDa (FIG. 2B).
[0228] Under reducing conditions, h15A7H V2-V3, V4-V2 and LH10
g4pFc showed a single band with the expected molecular weight of
around 75 kDa, whereas both h15A7H scFv.sub.2-LC and LC-scFv.sub.2
showed two major bands with similar molecular weight around 50 kDa
(FIG. 2C). One band was the scFv-LC or LC-scFv fusion protein, and
the other was the wild type h15A7H heavy chain. scFv.sub.4-crIgG4p
also showed two major bands, one representing the
scFv-CH1-hinge-CH2-CH3 (around 62.5 kDa) fusion protein, and the
other the scFv-kappa-fusion (around 37.5 kDa) protein (FIG. 2C). As
control, the h15A7H gave a single band with an expected molecular
weight of 150 kDa in the non-reducing gels (FIGS. 2A & 2B) and
two major bands (heavy chain: 50 kDa, light chain: 25 kDa) under
reducing conditions (FIG. 2C).
Binding of Humanized 15A711 Tetravalent Antibody Variants to
SP2/O-hPSGL-1 and SP2/O
[0229] The binding ability of h15A7H tetravalent antibodies was
evaluated in human PSGL-1 SP2/O cells. The h15A7H tetravalent
antibody bound positively to the SP2/O-hPSGL-1, but not to parental
SP2/O cell lacking of hPSGL-1 antigen (Table A below).
Additionally, wild type h15A7H and all of h15A7H tetravalent
antibodies gave similar binding activity on SP2/O-hPSGL-1 (Table
A). These results demonstrated that h15A7H tetravalent antibodies
retained binding reactivity to hPSGL-1 molecule.
TABLE-US-00003 TABLE A Binding activity (measured by mean
florescence intensity) of humanized 15A7H tetravalent antibodies to
SP2/O-hPSGL-1 and SP2/O. SP2/O-hPSGL-1 SP2/O (.mu.g/mL) 3 1 0.3 0.1
3 1 0.3 0.1 h15A7H 4667 6400 5943 3410 17 26 8 8 h15A7H LH10-g4pFc
4627 6677 5410 2902 19 18 31 30 h15A7H V2-V3-g4pFc 4535 6260 5731
3156 33 22 26 17 h15A7H scFv.sub.2-LC-IgG4p 4382 5744 7060 5543 24
12 11 24 h15A7H V4-V2-g4pFc 4923 6779 6454 3953 23 20 21 14 h15A7H
scFv.sub.4-crIgG4p 5938 6013 4637 2640 30 28 18 8 h15A7H
LC-scFv.sub.2-IgG4p 6026 5822 3477 3042 24 23 25 3 hIgG4p (control)
28 ND ND ND 33 ND ND ND ND: not done
In Vitro Apoptosis of SP2/O-hPSGL-1 Cells Induced by Humanized
15A711 Tetravalent Antibodies
[0230] Induction of apoptosis was evaluated by staining of Annexin
V and/or PI in SP2/O-hPSGL-1 cells after incubation with h15A7H or
tetravalent antibody. As shown in Table B below, the parental
antibody, h15A7H, did not induce apoptosis in SP2/O-hPSGL-1 cells
at the tested concentration of 0.5 and 0.0625 .mu.g/mL in the
absence of cross-linker. At the tested concentrations of 0.5
.mu.g/mL, all of the h15A7H tetravalent antibodies induced
apoptosis (ranging from 18-36%). At the lowest tested concentration
tested (0.0625 .mu.g/mL), 3 out of 6 tetravalent h15A7H antibodies,
LH10-g4pFc, V2-V3-g4pFc, and scFv.sub.2-LC-IgG4p, induced apoptosis
in 12-16% of cells, whereas h15A7H V4-V2-g4pFc, scFv.sub.4-crIgG4p,
and LC-scFv.sub.2-IgG4p did not induce cell death in SP2/O-hPSGL-1
at this lower dose. These data clearly demonstrate that all of the
h15A7H tetravalent antibodies possess apoptosis-inducing ability,
but that some tetravalent antibodies do so with greater
potency.
TABLE-US-00004 TABLE B In vitro apoptosis of SP2/O-hPSGL-1 cells
induced by humanized 15A7H tetravalent antibodies. Apoptosis % 0.5
.mu.g/mL 0.0625 .mu.g/mL (substrate background, n = 4) mean SD mean
SD h15A7H 2.75 3.95 1.5 3.32 h15A7H LH10-g4pFc 26.75 11.32 11.75*
5.50 h15A7H V2-V3-g4pFc 26.5 5.69 13.5* 5.45 h15A7H
scFv.sub.2-LC-IgG4p 23.75 9.00 15.5* 5.69 h15A7H V4-V2-g4pFc 30
4.55 0.5 3.00 h15A7H scFv.sub.4-crIgG4p 35.75 7.63 1.75 2.87 h15A7H
LC-scFv.sub.2-IgG4p 18 9.83 0.5 4.20 SD: standard deviation *T-test
P value < 0.05 (compared to treatment with V4-V2-g4pFc,
scFv.sub.4-crIgG4p and LC-scFv.sub.2-IgG4p).
Efficacy of h15A7H and h15A7H Tetravalent Antibodies in the
Inhibition of Trans-Vivo DTH Response in B6 Mice
[0231] The h15A7H and h15A7H tetravalent antibodies described above
were tested for their efficacy in the inhibition of trans vivo DTH
response in B6 mice. h15A7H antibody was intravenously injected
into mice at the doses of 10 and 1 mg/kg, whereas tetravalent
antibodies were intravenously injected into mice at the doses of 1
and 0.3 mg/kg. Experiments were conducted using PBMCs from four
different donors, and % inhibition was calculated to evaluate the
in vivo inhibitory efficacy.
[0232] As shown in Table C below, h15A7H antibody could inhibit
footpad swelling by a mean of 93% at the dose of 10 mg/kg. The
inhibition effect was reduced to 23% at the low dose of 1 mg/kg. As
for 15A7H tetravalent antibodies, variants such as h15A7H LH10-g4p
Fc, V2-V3-g4pFc and scFv.sub.2-LC-IgG4p remained effective in
inhibition even at doses of 1 or 0.3 mg/kg (with 59-76%
inhibition).
TABLE-US-00005 TABLE C Effect of h15A7H and h15A7H tetravalent
antibodies on Trans-vivo DTH. Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean SEM
% inhibition at 10 mg/kg h15A7H 71 104 82 116 93 10.2 % inhibition
at 1 mg/kg h15A7H 15 28 18 32 23 4.0 h15A7H LH10-g4pFc 75 51 109 69
76 12.1 h15A7H V2-V3-g4pFc 29 33 100 91 63 18.7 h15A7H
scFv.sub.2-LC-IgG4p 53 34 104 93 71 16.3 h15A7H V4-V2-g4pFc ND ND
11 44 27 16.5 h15A7H scFv.sub.4-crIgG4p 14 2 -18 6 1 6.8 h15A7H
LC-scFv.sub.2-IgG4p -17 16 14 29 10 9.8 % inhibition at 0.3 mg/kg
h15A7H LH10-g4pFc 28 73 109 50 65 17.2 h15A7H V2-V3-g4pFc 24 47 127
80 69 22.3 h15A7H scFv.sub.2-LC-IgG4p 74 24 66 73 59 11.8 h15A7H
V4-V2-g4pFc ND ND -16 11 -2 13.7 h15A7H scFv.sub.4-crIgG4p 2 9 -7 6
3 3.6 h15A7H LC-scFv.sub.2-IgG4p -4 19 5 15 9 5.2 ND: not done.;
SEM: the standard error of the mean
[0233] Plasma levels of h15A7H and h15A7H tetravalent antibodies
were also measured 24 hrs after i.v. administration (Table D). All
of the antibodies showed plasma levels around 6513-9025 ng/mL at 1
mg/kg except for V4-V2-g4pFc, which was undetectable after 24 hrs
circulation in vivo. Without wishing to be bound by theory, it is
thought that these results may indicate that the difference in
efficacy among h15A7H and tetravalent variants could be mainly due
to the differences in apoptosis-inducing ability, as demonstrated
in Table B.
TABLE-US-00006 TABLE D Plasma concentrations of h15A7H and h15A7H
tetravalent antibodies in B6 mice. Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean
SEM Conc. (ng/mL) at 10 mg/kg h15A7H 103411 100189 104471 110820
104723 2227 Conc. (ng/mL) at 1 mg/kg h15A7H 9087 8578 8316 10118
9025 398 h15A7H LH10-g4pFc 5698 7333 6335 6686 6513 508 h15A7H
V2-V3-g4pFc 6488 8173 6982 6478 7030 576 h15A7H scFv.sub.2-LC-IgG4p
5766 7082 7452 5979 6570 786 h15A7H V4-V2-g4pFc -- -- BLQ BLQ -- --
(<100) (<100) h15A7H scFv.sub.4-crIgG4p 6156 6924 5997 6353
6358 292 h15A7H LC-scFv.sub.2-IgG4p 7323 8432 9014 9006 8444 535
Conc. (ng/mL) at 0.3 mg/kg h15A7H LH10-g4pFc 1419 1853 1906 1793
1743 160 h15A7H V2-V3-g4pFc 1567 2284 2202 2065 2029 239 h15A7H
scFv.sub.2-LC-IgG4p 1344 1968 2112 1632 1764 241 h15A7H V4-V2-g4pFc
-- -- BLQ BLQ -- -- (<100) (<100) h15A7H scFv.sub.4-crIgG4p
1256 1909 1772 1668 1651 205 h15A7H LC-scFv.sub.2-IgG4p 1765 2493
2325 2356 2235 225 BLQ: beneath limit of quantification; SEM: the
standard error of the mean
[0234] In summary, these data demonstrate that various h15A7H
tetravalent antibodies possess differential abilities in induction
of apoptosis in vitro, which correlate with differential abilities
in the inhibition of a DTH response in trans vivo DTH murine model.
Those tetravalent antibodies with higher potency for apoptosis
induction showed enhanced efficacy compared to h15A7H in the
trans-vivo DTH model. These results suggest that some of these
h15A7H tetravalent variants may have potential advantages over
h15A7H for further clinical development.
[0235] Although the foregoing embodiments have been described in
some detail by way of illustration and example for purposes of
clarity of understanding, the descriptions and examples should not
be construed as limiting the scope of the present disclosure.
TABLE-US-00007 SEQUENCES All polypeptide sequences are presented
N-terminal to C-terminal unless otherwise noted. All polynucleotide
sequences are presented 5' to 3' unless otherwise noted. The three
CDRs in each chain are underlined, and the linker regions are shown
in lower case letters. Amino acid sequence of h15A7H LH10-g4pFc
(SEQ ID NO: 1)
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGT-
HFT
LTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTF-
SSF
GMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGA-
MDY
WGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYF-
EWY
QQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKggg-
gsg
gggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTI-
SRD
NAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsaaaESKYGPPCPPCPAPEFLGG-
PSV
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL-
NGK
EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY-
KTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK cDNA
sequence of h15A7H LH10-g4pFc (SEQ ID NO: 2)
GACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATC-
TAG
TCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGC-
TTC
TCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTC-
ACC
CTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCAC-
GTT
CGGTCAAGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctctGAAGTGCAACTGGTGG-
AGT
CTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGC-
TTT
GGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTAC-
CAT
CTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAA-
TGA
ATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGAC-
TAT
TGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcgg-
atc
cggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTG-
TAG
GGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGG-
TAC
CAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAG-
GTT
TAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATT-
ACT
GTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAAggtggaggcggttca-
ggc
ggaggtggctctGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTC-
CTG
TGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGT-
GGG
TCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGA-
GAT
AATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAG-
ATA
TGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggaggcggag-
gtt
ccgcggccgcaGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCA-
GTC
TTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGA-
CGT
GAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGC-
CGC
GGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGC-
AAG
GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA-
GCC
CCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC-
TGG
TCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC-
ACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGA-
GGG
GAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTC-
TGG GTAAATGA Amino acid sequence of h15A7H V2-V3-g4pFc (SEQ ID NO:
3)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN
TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQ-
SPS
SLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSL-
QPE
DFATYYCFQGSYVPLTFGCGTKVEIKastgsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQ-
KPG
KAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKggggsggg-
gsg
gggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFY-
ANA
VKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsaaaESKYGPPCP-
PCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS-
VLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW-
ESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
cDNA sequence of h15A7H V2-V3-g4pFc (SEQ ID NO: 4)
GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTC-
TGG
ATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACA-
TTA
ATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAG-
AAC
ACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTA-
CGG
AGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggag-
gtg
gctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCG-
AGC
TCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGG-
AAA
CACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGAT-
TTT
CTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCG-
GAG
GATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAAT-
CAA
AgcttcaaccggttcaGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCA-
CTA
TCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCA-
GGA
AAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGG-
GTC
TGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTT-
CAT
ATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctct-
ggc
ggtggcggatccggaggcggaggttccggaggtggcggaagtGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTT-
AGT
GCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGG-
TTC
GCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAAC-
GCA
GTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGC-
TGA
GGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCA-
CCC
TGGTCACAGTCTCCTCAggaggcggaggttccgcggccgcaGAGTCCAAATATGGTCCCCCATGCCCACCATGC-
CCA
GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCG-
GAC
CCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATG-
GCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTC-
ACC
GTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT-
CGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGA-
TGA
CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC-
AAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAG-
GCT
AACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC-
ACT ACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA Amino acid sequence of
h15A7H V4-V2-g4pFc (SEQ ID NO: 5)
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGT-
HFT
LTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKggggsggggsggggsggggsggggsEVQLVESGGGLVQP-
GGS
LRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDT-
AVY
YCARYASYGGGAMDYWGQGTLVTVSSastgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGK-
CLE
WVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSS-
ggg
gsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLL-
IYK
VSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKggggsaaaESKYGPPCP-
PCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS-
VLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW-
ESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
cDNA sequence of h15A7H V4-V2-g4pFc (SEQ ID NO: 6)
GACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATC-
TAG
TCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGC-
TTC
TCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTC-
ACC
CTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCAC-
GTT
CGGTTGTGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctctggcggtggcggatccg-
gag
gcggaggttccggaggtggcggaagtGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGA-
AGC
TTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGG-
GAA
GTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGAT-
TCA
CCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTG-
TAT
TACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTC-
CTC
AgcttcaaccggttcaGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGAC-
TCT
CCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTC-
GAG
TGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTC-
CAG
AGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTG-
CAA
GATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtgga-
ggc
ggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCA-
GAT
GACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCA-
TTG
TACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTAT-
AAA
GTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCAT-
CTC
TTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTG-
GCA
CCAAGGTGGAAATCAAAggaggcggaggttccgcggccgcaGAGTCCAAATATGGTCCCCCATGCCCACCATGC-
CCA
GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCG-
GAC
CCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATG-
GCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTC-
ACC
GTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT-
CGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGA-
TGA
CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC-
AAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAG-
GCT
AACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC-
ACT ACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA Amino acid sequence of
h15A7H scFv.sub.2-LC-IgG4p Light chain (SEQ ID NO: 7)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN
TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQ-
SPS
SLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSL-
QPE
DFATYYCFQGSYVPLTFGCGTKVEIKastgsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYF-
EWY
QQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKRTV-
AAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE-
KHK VYACEVTHQGLSSPVTKSFNRGEC cDNA sequence of h15A7H
scFv.sub.2-LC-IgG4p Light chain (SEQ ID NO: 8)
GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTC-
TGG
ATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACA-
TTA
ATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAG-
AAC
ACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTA-
CGG
AGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggag-
gtg
gctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCG-
AGC
TCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGG-
AAA
CACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGAT-
TTT
CTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCG-
GAG
GATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAAT-
CAA
AgcttcaaccggttcaggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTG-
TAG
GGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGG-
TAC
CAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAG-
GTT
TAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATT-
ACT
GTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAACGAACTGTGGCTGCA-
CCA
TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA-
CTT
CTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA-
CAG
AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC-
AAA
GTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA-
G Amino acid sequence of h15A7H LC- scFv.sub.2-IgG4p light chain
(SEQ ID NO: 9)
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGT-
HFT
LTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV-
QWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECggggsgggg-
sEV
QLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAK-
NTL
YLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSP-
SSL
SASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQP-
EDF ATYYCFQGSYVPLTFGCGTKVEIKAAAHHHHHHHHHH cDNA sequence of h15A7H
LC- scFv.sub.2-IgG4p light chain (SEQ ID NO: 10)
GACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATC-
TAG
TCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGC-
TTC
TCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTC-
ACC
CTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCAC-
GTT
CGGTCAAGGCACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG-
AGC
AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG-
AAG
GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAG-
CCT
CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG-
GCC
TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTggtggaggcggttcaggcggaggtggctctGAA-
GTG
CAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATT-
CAC
TTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATG-
GTG
GCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACC-
CTG
TACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGG-
GGG
TGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggct-
ctg
gcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCT-
TTG
TCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACAC-
CTA
TTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTG-
GTG
TCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGAT-
TTC
GCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAGC-
GGC CGCACATCATCATCATCATCACCACCACCACCACTAG Amino acid sequence of
h15A7H scFv.sub.2-LC-IgG4p and h15A7 LC- ScFv.sub.2 -IgG4p heavy
chain (SEQ ID NO: 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN
TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD-
YFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP-
CPA
PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV-
LTV
LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE-
SNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
cDNA sequence of h15A7H scFv.sub.2-LC-IgG4p and h15A7 LC-
scFv.sub.2 -IgG4p heavy chain (SEQ ID NO: 12)
GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTC-
TGG
ATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACA-
TTA
ATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAG-
AAC
ACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTA-
CGG
AGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAGCTTCCACCAAGGGCCCATCCG-
TCT
TCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTC-
CCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC-
CTC
AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACG-
TAG
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCA-
GCA
CCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGAC-
CCC
TGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG-
TGG
AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACC-
GTC
CTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGA-
GAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGA-
CCA
AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT-
GGG
CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCT-
AAC
CGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT-
ACA CACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA Amino acid sequence of
h15A7H scFv.sub.4-crIgG4p light chain (SEQ ID NO: 13)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN
TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQ-
SPS
SLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSL-
QPE
DFATYYCFQGSYVPLTFGCGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ-
SGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC cDNA
sequence of h15A7H scFv.sub.4-crIgG4p light chain (SEQ ID NO: 14)
GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTC-
TGG
ATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACA-
TTA
ATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAG-
AAC
ACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTA-
CGG
AGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggag-
gtg
gctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCG-
AGC
TCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGG-
AAA
CACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGAT-
TTT
CTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCG-
GAG
GATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAAT-
CAA
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG-
TTG
TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT-
AAC
TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA-
AGC
AGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT-
TCA ACAGGGGAGAGTGTTAG Amino acid sequence of h15A7H
scFv.sub.4-crIgG4p heavy chain (SEQ ID NO: 15)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN
TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQ-
SPS
SLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSL-
QPE
DFATYYCFQGSYVPLTFGCGTKVEIKASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS-
GVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP-
KPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK-
VSN
KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD-
SDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK cDNA sequence of
h15A7H scFv.sub.4-crIgG4p heavy chain (SEQ ID NO: 16)
GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTC-
TGG
ATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACA-
TTA
ATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAG-
AAC
ACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTA-
CGG
AGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggag-
gtg
gctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCG-
AGC
TCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGG-
AAA
CACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGAT-
TTT
CTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCG-
GAG
GATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAAT-
CAA
AGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCC-
TGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG-
CAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT-
GGG
CACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAAT-
ATG
GTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCC-
AAG
GACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGT-
CCA
GTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCA-
CGT
ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCC-
AAC
AAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACAC-
CCT
GCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCG-
ACA
TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC-
GGC
TCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGT-
GAT GCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA
Amino acid sequence of h15A7H CDR-H1 (SEQ ID NO: 17) SFGMH Amino
acid sequence of h15A7H CDR-H2 (SEQ ID NO: 18) YINGGSSTIFYANAVKG
Amino acid sequence of h15A7H CDR-H3 (SEQ ID NO: 19) YASYGGGAMDY
Amino acid sequence of h15A7H CDR-L1 (SEQ ID NO: 20)
RSSQSIVHNDGNTYFE Amino acid sequence of h15A7H CDR-L2 (SEQ ID NO:
21) KVSNRFS Amino acid sequence of h15A7H CDR-L3 (SEQ ID NO: 22)
FQGSYVPLT Amino acid sequence of h15A7H VH (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSS Amino acid sequence
of h15A7H VL (SEQ ID NO: 24)
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGT-
HFT LTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIK Amino acid sequence of
linker sequence repeat (SEQ ID NO: 25) ggggs Amino acid sequence of
linker with Fc (SEQ ID NO: 26) ggggsaaa Amino acid sequence of taFv
linker (SEQ ID NO: 27) astgs Amino acid sequence of scFv light
chain linker (SEQ ID NO: 28) astgsggggs Amino acid sequence of
h15A7H VH G44C(SEQ ID NO: 29)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDN-
AKN TLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSS Amino acid sequence
of h15A7H VL Q100C(SEQ ID NO: 30)
DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGT-
HFT LTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIK Amino acid sequence of
human PSGL-1 (SEQ ID NO: 31)
MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEMLRNSTDTT
PLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAMEIQTTQPAATEAQTTQPVPTE
AQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAATEAQTTQPTGLEAQTTAPAAMEAQTTAPAAME
AQTTPPAAMEAQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKR
GLFIPFSVSSVTHKGIPMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGH
MYPVRNYSPTEMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP
Amino acid sequence of shorter human PSGL-1 variant (SEQ ID NO: 32)
MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEMLRNSTDTT
PLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAMEIQTTQPAATEAQTTPLAATE
AQTTRLTATEAQTTPLAATEAQTTPPAATEAQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAAME
AQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKRGLFIPFSVSS
VTHKGIPMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGHMYPVRNYSPT
EMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP
Sequence CWU 1
1
361746PRTArtificial SequenceSynthetic Construct 1Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Asn 20 25 30Asp Gly
Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile65
70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln
Gly 85 90 95Ser Tyr Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Gln Leu Val Glu 115 120 125Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys 130 135 140Ala Ala Ser Gly Phe Thr Phe Ser
Ser Phe Gly Met His Trp Val Arg145 150 155 160Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ala Tyr Ile Asn Gly Gly 165 170 175Ser Ser Thr
Ile Phe Tyr Ala Asn Ala Val Lys Gly Arg Phe Thr Ile 180 185 190Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 195 200
205Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ala Ser Tyr
210 215 220Gly Gly Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val225 230 235 240Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly 245 250 255Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Asp Ile Gln Met Thr 260 265 270Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile 275 280 285Thr Cys Arg Ser Ser
Gln Ser Ile Val His Asn Asp Gly Asn Thr Tyr 290 295 300Phe Glu Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile305 310 315
320Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
325 330 335Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 340 345 350Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser
Tyr Val Pro Leu 355 360 365Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Gly Gly Gly Gly Ser 370 375 380Gly Gly Gly Gly Ser Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu385 390 395 400Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 405 410 415Thr Phe Ser
Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 420 425 430Gly
Leu Glu Trp Val Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe 435 440
445Tyr Ala Asn Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
450 455 460Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr465 470 475 480Ala Val Tyr Tyr Cys Ala Arg Tyr Ala Ser Tyr
Gly Gly Gly Ala Met 485 490 495Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly 500 505 510Gly Ser Ala Ala Ala Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys 515 520 525Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 530 535 540Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys545 550 555
560Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
565 570 575Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 580 585 590Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 595 600 605His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 610 615 620Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly625 630 635 640Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 645 650 655Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 660 665 670Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 675 680
685Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
690 695 700Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
Gly Asn705 710 715 720Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 725 730 735Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 740 74522241DNAArtificial SequenceSynthetic Construct
2gacattcaga tgacccaatc tccgagctct ttgtctgcgt ctgtagggga tagggtcact
60atcacctgca gatctagtca gagcattgta cataatgatg gaaacaccta ttttgaatgg
120taccaacaga aaccaggaaa ggcacccaag cttctcatct ataaagtttc
caatcgattt 180tctggtgtcc catccaggtt tagtggcagt gggtctggga
cacacttcac cctcaccatc 240tcttctctgc agccggagga tttcgcaacc
tattactgtt ttcaaggttc atatgttcct 300ctcacgttcg gtcaaggcac
caaggtggaa atcaaaggtg gaggcggttc aggcggaggt 360ggctctgaag
tgcaactggt ggagtctggg ggaggcttag tgcagcctgg aggaagcttg
420agactctcct gtgcagcctc tggattcact ttcagtagct ttggaatgca
ctgggttcgc 480caggctccag ggaagggact cgagtgggtc gcatacatta
atggtggcag tagtaccatc 540ttctatgcaa acgcagtgaa gggccgattc
accatctcca gagataatgc caagaacacc 600ctgtacctgc aaatgaattc
tctgagggct gaggacacgg ccgtgtatta ctgtgcaaga 660tatgctagtt
acggaggggg tgctatggac tattggggcc aaggcaccct ggtcacagtc
720tcctcaggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc
cggaggcgga 780ggttccggag gtggcggaag tgacattcag atgacccaat
ctccgagctc tttgtctgcg 840tctgtagggg atagggtcac tatcacctgc
agatctagtc agagcattgt acataatgat 900ggaaacacct attttgaatg
gtaccaacag aaaccaggaa aggcacccaa gcttctcatc 960tataaagttt
ccaatcgatt ttctggtgtc ccatccaggt ttagtggcag tgggtctggg
1020acacacttca ccctcaccat ctcttctctg cagccggagg atttcgcaac
ctattactgt 1080tttcaaggtt catatgttcc tctcacgttc ggtcaaggca
ccaaggtgga aatcaaaggt 1140ggaggcggtt caggcggagg tggctctgaa
gtgcaactgg tggagtctgg gggaggctta 1200gtgcagcctg gaggaagctt
gagactctcc tgtgcagcct ctggattcac tttcagtagc 1260tttggaatgc
actgggttcg ccaggctcca gggaagggac tcgagtgggt cgcatacatt
1320aatggtggca gtagtaccat cttctatgca aacgcagtga agggccgatt
caccatctcc 1380agagataatg ccaagaacac cctgtacctg caaatgaatt
ctctgagggc tgaggacacg 1440gccgtgtatt actgtgcaag atatgctagt
tacggagggg gtgctatgga ctattggggc 1500caaggcaccc tggtcacagt
ctcctcagga ggcggaggtt ccgcggccgc agagtccaaa 1560tatggtcccc
catgcccacc atgcccagca cctgagttcc tggggggacc atcagtcttc
1620ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga
ggtcacgtgc 1680gtggtggtgg acgtgagcca ggaagacccc gaggtccagt
tcaactggta cgtggatggc 1740gtggaggtgc ataatgccaa gacaaagccg
cgggaggagc agttcaacag cacgtaccgt 1800gtggtcagcg tcctcaccgt
cctgcaccag gactggctga acggcaagga gtacaagtgc 1860aaggtctcca
acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg
1920cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat
gaccaagaac 1980caggtcagcc tgacctgcct ggtcaaaggc ttctacccca
gcgacatcgc cgtggagtgg 2040gagagcaatg ggcagccgga gaacaactac
aagaccacgc ctcccgtgct ggactccgac 2100ggctccttct tcctctacag
caggctaacc gtggacaaga gcaggtggca ggaggggaat 2160gtcttctcat
gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc
2220tccctgtctc tgggtaaatg a 22413756PRTArtificial SequenceSynthetic
Construct 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys
Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe
Tyr Ala Asn Ala Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Ala Ser Tyr Gly
Gly Gly Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val145 150
155 160Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val
His 165 170 175Asn Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys
Pro Gly Lys 180 185 190Ala Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser Gly Val 195 200 205Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr His Phe Thr Leu Thr 210 215 220Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Phe Gln225 230 235 240Gly Ser Tyr Val
Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu Ile 245 250 255Lys Ala
Ser Thr Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 260 265
270Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser
275 280 285Gln Ser Ile Val His Asn Asp Gly Asn Thr Tyr Phe Glu Trp
Tyr Gln 290 295 300Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Lys Val Ser Asn305 310 315 320Arg Phe Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr 325 330 335His Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr 340 345 350Tyr Tyr Cys Phe Gln
Gly Ser Tyr Val Pro Leu Thr Phe Gly Gln Gly 355 360 365Thr Lys Val
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 370 375 380Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu385 390
395 400Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser 405 410 415Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Phe Gly 420 425 430Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ala 435 440 445Tyr Ile Asn Gly Gly Ser Ser Thr Ile
Phe Tyr Ala Asn Ala Val Lys 450 455 460Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr Leu465 470 475 480Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 485 490 495Arg Tyr
Ala Ser Tyr Gly Gly Gly Ala Met Asp Tyr Trp Gly Gln Gly 500 505
510Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ala Ala Ala Glu
515 520 525Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe Leu 530 535 540Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu545 550 555 560Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 565 570 575Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 580 585 590Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 595 600 605Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 610 615 620Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser625 630
635 640Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln 645 650 655Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val 660 665 670Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val 675 680 685Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro 690 695 700Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr705 710 715 720Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 725 730 735Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 740 745
750Ser Leu Gly Lys 75542271DNAArtificial SequenceSynthetic
Construct 4gaagtgcaac tggtggagtc tgggggaggc ttagtgcagc ctggaggaag
cttgagactc 60tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt
tcgccaggct 120ccagggaagt gtctcgagtg ggtcgcatac attaatggtg
gcagtagtac catcttctat 180gcaaacgcag tgaagggccg attcaccatc
tccagagata atgccaagaa caccctgtac 240ctgcaaatga attctctgag
ggctgaggac acggccgtgt attactgtgc aagatatgct 300agttacggag
ggggtgctat ggactattgg ggccaaggca ccctggtcac agtctcctca
360ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatccggagg
cggaggttcc 420ggaggtggcg gaagtgacat tcagatgacc caatctccga
gctctttgtc tgcgtctgta 480ggggataggg tcactatcac ctgcagatct
agtcagagca ttgtacataa tgatggaaac 540acctattttg aatggtacca
acagaaacca ggaaaggcac ccaagcttct catctataaa 600gtttccaatc
gattttctgg tgtcccatcc aggtttagtg gcagtgggtc tgggacacac
660ttcaccctca ccatctcttc tctgcagccg gaggatttcg caacctatta
ctgttttcaa 720ggttcatatg ttcctctcac gttcggttgt ggcaccaagg
tggaaatcaa agcttcaacc 780ggttcagaca ttcagatgac ccaatctccg
agctctttgt ctgcgtctgt aggggatagg 840gtcactatca cctgcagatc
tagtcagagc attgtacata atgatggaaa cacctatttt 900gaatggtacc
aacagaaacc aggaaaggca cccaagcttc tcatctataa agtttccaat
960cgattttctg gtgtcccatc caggtttagt ggcagtgggt ctgggacaca
cttcaccctc 1020accatctctt ctctgcagcc ggaggatttc gcaacctatt
actgttttca aggttcatat 1080gttcctctca cgttcggtca aggcaccaag
gtggaaatca aaggtggagg cggttcaggc 1140ggaggtggct ctggcggtgg
cggatccgga ggcggaggtt ccggaggtgg cggaagtgaa 1200gtgcaactgg
tggagtctgg gggaggctta gtgcagcctg gaggaagctt gagactctcc
1260tgtgcagcct ctggattcac tttcagtagc tttggaatgc actgggttcg
ccaggctcca 1320gggaagggac tcgagtgggt cgcatacatt aatggtggca
gtagtaccat cttctatgca 1380aacgcagtga agggccgatt caccatctcc
agagataatg ccaagaacac cctgtacctg 1440caaatgaatt ctctgagggc
tgaggacacg gccgtgtatt actgtgcaag atatgctagt 1500tacggagggg
gtgctatgga ctattggggc caaggcaccc tggtcacagt ctcctcagga
1560ggcggaggtt ccgcggccgc agagtccaaa tatggtcccc catgcccacc
atgcccagca 1620cctgagttcc tggggggacc atcagtcttc ctgttccccc
caaaacccaa ggacactctc 1680atgatctccc ggacccctga ggtcacgtgc
gtggtggtgg acgtgagcca ggaagacccc 1740gaggtccagt tcaactggta
cgtggatggc gtggaggtgc ataatgccaa gacaaagccg 1800cgggaggagc
agttcaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag
1860gactggctga acggcaagga gtacaagtgc aaggtctcca acaaaggcct
cccgtcctcc 1920atcgagaaaa ccatctccaa agccaaaggg cagccccgag
agccacaggt gtacaccctg 1980cccccatccc aggaggagat gaccaagaac
caggtcagcc tgacctgcct ggtcaaaggc 2040ttctacccca gcgacatcgc
cgtggagtgg gagagcaatg ggcagccgga gaacaactac 2100aagaccacgc
ctcccgtgct ggactccgac ggctccttct tcctctacag caggctaacc
2160gtggacaaga gcaggtggca ggaggggaat gtcttctcat gctccgtgat
gcatgaggct 2220ctgcacaacc actacacaca gaagagcctc tccctgtctc
tgggtaaatg a 22715756PRTArtificial SequenceSynthetic Construct 5Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Asn
20 25 30Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly Lys
Ala 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr
Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Phe Gln Gly 85 90 95Ser Tyr Val Pro Leu Thr Phe Gly Cys Gly
Thr Lys Val Glu Ile Lys 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser 130 135 140Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala145 150 155 160Ala
Ser Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln 165 170
175Ala Pro Gly Lys Cys Leu Glu Trp Val Ala Tyr Ile Asn Gly Gly Ser
180 185
190Ser Thr Ile Phe Tyr Ala Asn Ala Val Lys Gly Arg Phe Thr Ile Ser
195 200 205Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg 210 215 220Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Tyr
Ala Ser Tyr Gly225 230 235 240Gly Gly Ala Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 245 250 255Ser Ala Ser Thr Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly 260 265 270Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 275 280 285Phe Thr Phe
Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly 290 295 300Lys
Cys Leu Glu Trp Val Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile305 310
315 320Phe Tyr Ala Asn Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn 325 330 335Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp 340 345 350Thr Ala Val Tyr Tyr Cys Ala Arg Tyr Ala Ser
Tyr Gly Gly Gly Ala 355 360 365Met Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Gly Gly 370 375 380Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly385 390 395 400Gly Ser Gly Gly
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 405 410 415Ser Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser 420 425
430Ser Gln Ser Ile Val His Asn Asp Gly Asn Thr Tyr Phe Glu Trp Tyr
435 440 445Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys
Val Ser 450 455 460Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly465 470 475 480Thr His Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala 485 490 495Thr Tyr Tyr Cys Phe Gln Gly
Ser Tyr Val Pro Leu Thr Phe Gly Cys 500 505 510Gly Thr Lys Val Glu
Ile Lys Gly Gly Gly Gly Ser Ala Ala Ala Glu 515 520 525Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 530 535 540Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu545 550
555 560Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 565 570 575Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu 580 585 590Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr 595 600 605Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 610 615 620Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser625 630 635 640Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 645 650 655Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 660 665
670Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
675 680 685Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro 690 695 700Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr705 710 715 720Val Asp Lys Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val 725 730 735Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 740 745 750Ser Leu Gly Lys
75562271DNAArtificial SequenceSynthetic Construct 6gacattcaga
tgacccaatc tccgagctct ttgtctgcgt ctgtagggga tagggtcact 60atcacctgca
gatctagtca gagcattgta cataatgatg gaaacaccta ttttgaatgg
120taccaacaga aaccaggaaa ggcacccaag cttctcatct ataaagtttc
caatcgattt 180tctggtgtcc catccaggtt tagtggcagt gggtctggga
cacacttcac cctcaccatc 240tcttctctgc agccggagga tttcgcaacc
tattactgtt ttcaaggttc atatgttcct 300ctcacgttcg gttgtggcac
caaggtggaa atcaaaggtg gaggcggttc aggcggaggt 360ggctctggcg
gtggcggatc cggaggcgga ggttccggag gtggcggaag tgaagtgcaa
420ctggtggagt ctgggggagg cttagtgcag cctggaggaa gcttgagact
ctcctgtgca 480gcctctggat tcactttcag tagctttgga atgcactggg
ttcgccaggc tccagggaag 540tgtctcgagt gggtcgcata cattaatggt
ggcagtagta ccatcttcta tgcaaacgca 600gtgaagggcc gattcaccat
ctccagagat aatgccaaga acaccctgta cctgcaaatg 660aattctctga
gggctgagga cacggccgtg tattactgtg caagatatgc tagttacgga
720gggggtgcta tggactattg gggccaaggc accctggtca cagtctcctc
agcttcaacc 780ggttcagaag tgcaactggt ggagtctggg ggaggcttag
tgcagcctgg aggaagcttg 840agactctcct gtgcagcctc tggattcact
ttcagtagct ttggaatgca ctgggttcgc 900caggctccag ggaagtgtct
cgagtgggtc gcatacatta atggtggcag tagtaccatc 960ttctatgcaa
acgcagtgaa gggccgattc accatctcca gagataatgc caagaacacc
1020ctgtacctgc aaatgaattc tctgagggct gaggacacgg ccgtgtatta
ctgtgcaaga 1080tatgctagtt acggaggggg tgctatggac tattggggcc
aaggcaccct ggtcacagtc 1140tcctcaggtg gaggcggttc aggcggaggt
ggctctggcg gtggcggatc cggaggcgga 1200ggttccggag gtggcggaag
tgacattcag atgacccaat ctccgagctc tttgtctgcg 1260tctgtagggg
atagggtcac tatcacctgc agatctagtc agagcattgt acataatgat
1320ggaaacacct attttgaatg gtaccaacag aaaccaggaa aggcacccaa
gcttctcatc 1380tataaagttt ccaatcgatt ttctggtgtc ccatccaggt
ttagtggcag tgggtctggg 1440acacacttca ccctcaccat ctcttctctg
cagccggagg atttcgcaac ctattactgt 1500tttcaaggtt catatgttcc
tctcacgttc ggttgtggca ccaaggtgga aatcaaagga 1560ggcggaggtt
ccgcggccgc agagtccaaa tatggtcccc catgcccacc atgcccagca
1620cctgagttcc tggggggacc atcagtcttc ctgttccccc caaaacccaa
ggacactctc 1680atgatctccc ggacccctga ggtcacgtgc gtggtggtgg
acgtgagcca ggaagacccc 1740gaggtccagt tcaactggta cgtggatggc
gtggaggtgc ataatgccaa gacaaagccg 1800cgggaggagc agttcaacag
cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1860gactggctga
acggcaagga gtacaagtgc aaggtctcca acaaaggcct cccgtcctcc
1920atcgagaaaa ccatctccaa agccaaaggg cagccccgag agccacaggt
gtacaccctg 1980cccccatccc aggaggagat gaccaagaac caggtcagcc
tgacctgcct ggtcaaaggc 2040ttctacccca gcgacatcgc cgtggagtgg
gagagcaatg ggcagccgga gaacaactac 2100aagaccacgc ctcccgtgct
ggactccgac ggctccttct tcctctacag caggctaacc 2160gtggacaaga
gcaggtggca ggaggggaat gtcttctcat gctccgtgat gcatgaggct
2220ctgcacaacc actacacaca gaagagcctc tccctgtctc tgggtaaatg a
22717486PRTArtificial SequenceSynthetic Construct 7Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45Ala
Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe Tyr Ala Asn Ala Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Tyr Ala Ser Tyr Gly Gly Gly Ala Met Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val145 150 155 160Gly Asp Arg Val Thr
Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His 165 170 175Asn Asp Gly
Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly Lys 180 185 190Ala
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val 195 200
205Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr
210 215 220Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln225 230 235 240Gly Ser Tyr Val Pro Leu Thr Phe Gly Cys Gly
Thr Lys Val Glu Ile 245 250 255Lys Ala Ser Thr Gly Ser Gly Gly Gly
Gly Ser Asp Ile Gln Met Thr 260 265 270Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile 275 280 285Thr Cys Arg Ser Ser
Gln Ser Ile Val His Asn Asp Gly Asn Thr Tyr 290 295 300Phe Glu Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile305 310 315
320Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
325 330 335Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 340 345 350Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser
Tyr Val Pro Leu 355 360 365Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 370 375 380Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly385 390 395 400Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 405 410 415Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 420 425 430Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 435 440
445Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
450 455 460Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser465 470 475 480Phe Asn Arg Gly Glu Cys 48581461DNAArtificial
SequenceSynthetic Construct 8gaagtgcaac tggtggagtc tgggggaggc
ttagtgcagc ctggaggaag cttgagactc 60tcctgtgcag cctctggatt cactttcagt
agctttggaa tgcactgggt tcgccaggct 120ccagggaagt gtctcgagtg
ggtcgcatac attaatggtg gcagtagtac catcttctat 180gcaaacgcag
tgaagggccg attcaccatc tccagagata atgccaagaa caccctgtac
240ctgcaaatga attctctgag ggctgaggac acggccgtgt attactgtgc
aagatatgct 300agttacggag ggggtgctat ggactattgg ggccaaggca
ccctggtcac agtctcctca 360ggtggaggcg gttcaggcgg aggtggctct
ggcggtggcg gatccggagg cggaggttcc 420ggaggtggcg gaagtgacat
tcagatgacc caatctccga gctctttgtc tgcgtctgta 480ggggataggg
tcactatcac ctgcagatct agtcagagca ttgtacataa tgatggaaac
540acctattttg aatggtacca acagaaacca ggaaaggcac ccaagcttct
catctataaa 600gtttccaatc gattttctgg tgtcccatcc aggtttagtg
gcagtgggtc tgggacacac 660ttcaccctca ccatctcttc tctgcagccg
gaggatttcg caacctatta ctgttttcaa 720ggttcatatg ttcctctcac
gttcggttgt ggcaccaagg tggaaatcaa agcttcaacc 780ggttcaggag
gtggcggaag tgacattcag atgacccaat ctccgagctc tttgtctgcg
840tctgtagggg atagggtcac tatcacctgc agatctagtc agagcattgt
acataatgat 900ggaaacacct attttgaatg gtaccaacag aaaccaggaa
aggcacccaa gcttctcatc 960tataaagttt ccaatcgatt ttctggtgtc
ccatccaggt ttagtggcag tgggtctggg 1020acacacttca ccctcaccat
ctcttctctg cagccggagg atttcgcaac ctattactgt 1080tttcaaggtt
catatgttcc tctcacgttc ggtcaaggca ccaaggtgga aatcaaacga
1140actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt
gaaatctgga 1200actgcctctg ttgtgtgcct gctgaataac ttctatccca
gagaggccaa agtacagtgg 1260aaggtggata acgccctcca atcgggtaac
tcccaggaga gtgtcacaga gcaggacagc 1320aaggacagca cctacagcct
cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 1380cacaaagtct
acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc
1440ttcaacaggg gagagtgtta g 14619499PRTArtificial SequenceSynthetic
Construct 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile
Val His Asn 20 25 30Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys
Pro Gly Lys Ala 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
His Phe Thr Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Phe Gln Gly 85 90 95Ser Tyr Val Pro Leu Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150
155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys Gly Gly Gly Gly Ser 210 215 220Gly Gly Gly Gly Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu225 230 235 240Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 245 250 255Thr Phe
Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 260 265
270Cys Leu Glu Trp Val Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe
275 280 285Tyr Ala Asn Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala 290 295 300Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr305 310 315 320Ala Val Tyr Tyr Cys Ala Arg Tyr Ala
Ser Tyr Gly Gly Gly Ala Met 325 330 335Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly 340 345 350Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 355 360 365Ser Gly Gly
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 370 375 380Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser385 390
395 400Gln Ser Ile Val His Asn Asp Gly Asn Thr Tyr Phe Glu Trp Tyr
Gln 405 410 415Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys
Val Ser Asn 420 425 430Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr 435 440 445His Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr 450 455 460Tyr Tyr Cys Phe Gln Gly Ser
Tyr Val Pro Leu Thr Phe Gly Cys Gly465 470 475 480Thr Lys Val Glu
Ile Lys Ala Ala Ala His His His His His His His 485 490 495His His
His101500DNAArtificial SequenceSynthetic Construct 10gacattcaga
tgacccaatc tccgagctct ttgtctgcgt ctgtagggga tagggtcact 60atcacctgca
gatctagtca gagcattgta cataatgatg gaaacaccta ttttgaatgg
120taccaacaga aaccaggaaa ggcacccaag cttctcatct ataaagtttc
caatcgattt 180tctggtgtcc catccaggtt tagtggcagt gggtctggga
cacacttcac cctcaccatc 240tcttctctgc agccggagga tttcgcaacc
tattactgtt ttcaaggttc atatgttcct 300ctcacgttcg gtcaaggcac
caaggtggaa atcaaacgaa ctgtggctgc accatctgtc 360ttcatcttcc
cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg
420ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa
cgccctccaa 480tcgggtaact cccaggagag tgtcacagag caggacagca
aggacagcac ctacagcctc 540agcagcaccc tgacgctgag caaagcagac
tacgagaaac acaaagtcta cgcctgcgaa 600gtcacccatc agggcctgag
ctcgcccgtc acaaagagct tcaacagggg agagtgtggt 660ggaggcggtt
caggcggagg tggctctgaa gtgcaactgg tggagtctgg gggaggctta
720gtgcagcctg gaggaagctt gagactctcc tgtgcagcct ctggattcac
tttcagtagc 780tttggaatgc actgggttcg ccaggctcca gggaagtgtc
tcgagtgggt cgcatacatt 840aatggtggca gtagtaccat cttctatgca
aacgcagtga agggccgatt caccatctcc 900agagataatg ccaagaacac
cctgtacctg caaatgaatt ctctgagggc tgaggacacg 960gccgtgtatt
actgtgcaag atatgctagt tacggagggg gtgctatgga ctattggggc
1020caaggcaccc tggtcacagt ctcctcaggt ggaggcggtt caggcggagg
tggctctggc 1080ggtggcggat ccggaggcgg aggttccgga ggtggcggaa
gtgacattca gatgacccaa 1140tctccgagct ctttgtctgc gtctgtaggg
gatagggtca ctatcacctg cagatctagt 1200cagagcattg tacataatga
tggaaacacc tattttgaat ggtaccaaca gaaaccagga 1260aaggcaccca
agcttctcat ctataaagtt tccaatcgat tttctggtgt cccatccagg
1320tttagtggca gtgggtctgg gacacacttc accctcacca tctcttctct
gcagccggag 1380gatttcgcaa cctattactg ttttcaaggt tcatatgttc
ctctcacgtt cggttgtggc 1440accaaggtgg aaatcaaagc ggccgcacat
catcatcatc atcaccacca ccaccactag 150011447PRTArtificial
SequenceSynthetic Construct 11Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe Tyr Ala
Asn Ala Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Ala Ser Tyr Gly Gly Gly
Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 445121344DNAArtificial SequenceSynthetic
Construct 12gaagtgcaac tggtggagtc tgggggaggc ttagtgcagc ctggaggaag
cttgagactc 60tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt
tcgccaggct 120ccagggaagg gactcgagtg ggtcgcatac attaatggtg
gcagtagtac catcttctat 180gcaaacgcag tgaagggccg attcaccatc
tccagagata atgccaagaa caccctgtac 240ctgcaaatga attctctgag
ggctgaggac acggccgtgt attactgtgc aagatatgct 300agttacggag
ggggtgctat ggactattgg ggccaaggca ccctggtcac agtctcctca
360gcttccacca agggcccatc cgtcttcccc ctggcgccct gctccaggag
cacctccgag 420agcacagccg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 540ggactctact ccctcagcag
cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 600tacacctgca
acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc
660aaatatggtc ccccatgccc accatgccca gcacctgagt tcctgggggg
accatcagtc 720ttcctgttcc ccccaaaacc caaggacact ctcatgatct
cccggacccc tgaggtcacg 780tgcgtggtgg tggacgtgag ccaggaagac
cccgaggtcc agttcaactg gtacgtggat 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagttcaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag
960tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc
caaagccaaa 1020gggcagcccc gagagccaca ggtgtacacc ctgcccccat
cccaggagga gatgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct
tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg
1260aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 1320ctctccctgt ctctgggtaa atga 134413364PRTArtificial
SequenceSynthetic Construct 13Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Gly Gly
Ser Ser Thr Ile Phe Tyr Ala Asn Ala Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Tyr Ala Ser Tyr Gly Gly Gly Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val145 150 155 160Gly Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser Ile Val His 165 170 175Asn Asp Gly Asn Thr Tyr Phe
Glu Trp Tyr Gln Gln Lys Pro Gly Lys 180 185 190Ala Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val 195 200 205Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr 210 215 220Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln225 230
235 240Gly Ser Tyr Val Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu
Ile 245 250 255Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp 260 265 270Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn 275 280 285Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu 290 295 300Gln Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp305 310 315 320Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 325 330 335Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 340 345
350Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 355
360141095DNAArtificial SequenceSynthetic Construct 14gaagtgcaac
tggtggagtc tgggggaggc ttagtgcagc ctggaggaag cttgagactc 60tcctgtgcag
cctctggatt cactttcagt agctttggaa tgcactgggt tcgccaggct
120ccagggaagt gtctcgagtg ggtcgcatac attaatggtg gcagtagtac
catcttctat 180gcaaacgcag tgaagggccg attcaccatc tccagagata
atgccaagaa caccctgtac 240ctgcaaatga attctctgag ggctgaggac
acggccgtgt attactgtgc aagatatgct 300agttacggag ggggtgctat
ggactattgg ggccaaggca ccctggtcac agtctcctca 360ggtggaggcg
gttcaggcgg aggtggctct ggcggtggcg gatccggagg cggaggttcc
420ggaggtggcg gaagtgacat tcagatgacc caatctccga gctctttgtc
tgcgtctgta 480ggggataggg tcactatcac ctgcagatct agtcagagca
ttgtacataa tgatggaaac 540acctattttg aatggtacca acagaaacca
ggaaaggcac ccaagcttct catctataaa 600gtttccaatc gattttctgg
tgtcccatcc aggtttagtg gcagtgggtc tgggacacac 660ttcaccctca
ccatctcttc tctgcagccg gaggatttcg caacctatta ctgttttcaa
720ggttcatatg ttcctctcac gttcggttgt ggcaccaagg tggaaatcaa
acgaactgtg 780gctgcaccat ctgtcttcat cttcccgcca tctgatgagc
agttgaaatc tggaactgcc 840tctgttgtgt gcctgctgaa taacttctat
cccagagagg ccaaagtaca gtggaaggtg 900gataacgccc tccaatcggg
taactcccag gagagtgtca cagagcagga cagcaaggac 960agcacctaca
gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa
1020gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa
gagcttcaac 1080aggggagagt gttag 109515584PRTArtificial
SequenceSynthetic Construct 15Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Gly Gly
Ser Ser Thr Ile Phe Tyr Ala Asn Ala Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Tyr Ala Ser Tyr Gly Gly Gly Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val145 150 155 160Gly Asp Arg Val Thr Ile Thr Cys Arg
Ser Ser Gln Ser Ile Val His 165 170 175Asn Asp Gly Asn Thr Tyr Phe
Glu Trp Tyr Gln Gln Lys Pro Gly Lys 180 185 190Ala Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val 195 200 205Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr 210 215 220Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln225 230
235 240Gly Ser Tyr Val Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu
Ile 245 250 255Lys Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser 260 265 270Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp 275 280 285Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr 290 295 300Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr305 310 315 320Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 325 330 335Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 340 345
350Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
355 360 365Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 370 375 380Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val385 390 395 400Val Asp Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val 405 410 415Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 420 425 430Phe Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 435 440 445Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 450 455 460Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro465 470
475 480Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr 485 490 495Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser 500 505 510Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 515 520 525Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 530 535 540Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe545 550 555 560Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 565 570 575Ser Leu
Ser Leu Ser Leu Gly Lys 580161755DNAArtificial SequenceSynthetic
Construct 16gaagtgcaac tggtggagtc tgggggaggc ttagtgcagc ctggaggaag
cttgagactc 60tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt
tcgccaggct 120ccagggaagt gtctcgagtg ggtcgcatac attaatggtg
gcagtagtac catcttctat 180gcaaacgcag tgaagggccg attcaccatc
tccagagata atgccaagaa caccctgtac 240ctgcaaatga attctctgag
ggctgaggac acggccgtgt attactgtgc aagatatgct 300agttacggag
ggggtgctat ggactattgg ggccaaggca ccctggtcac agtctcctca
360ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatccggagg
cggaggttcc 420ggaggtggcg gaagtgacat tcagatgacc caatctccga
gctctttgtc tgcgtctgta 480ggggataggg tcactatcac ctgcagatct
agtcagagca ttgtacataa tgatggaaac 540acctattttg aatggtacca
acagaaacca ggaaaggcac ccaagcttct catctataaa 600gtttccaatc
gattttctgg tgtcccatcc aggtttagtg gcagtgggtc tgggacacac
660ttcaccctca ccatctcttc tctgcagccg gaggatttcg caacctatta
ctgttttcaa 720ggttcatatg ttcctctcac gttcggttgt ggcaccaagg
tggaaatcaa agcttccacc 780aagggcccat ccgtcttccc cctggcgccc
tgctccagga gcacctccga gagcacagcc 840gccctgggct gcctggtcaa
ggactacttc cccgaaccgg tgacggtgtc gtggaactca 900ggcgccctga
ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac
960tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac
ctacacctgc 1020aacgtagatc acaagcccag caacaccaag gtggacaaga
gagttgagtc caaatatggt 1080cccccatgcc caccatgccc agcacctgag
ttcctggggg gaccatcagt cttcctgttc 1140cccccaaaac ccaaggacac
tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 1200gtggacgtga
gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag
1260gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta
ccgtgtggtc 1320agcgtcctca ccgtcctgca ccaggactgg ctgaacggca
aggagtacaa gtgcaaggtc 1380tccaacaaag gcctcccgtc ctccatcgag
aaaaccatct ccaaagccaa agggcagccc 1440cgagagccac aggtgtacac
cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1500agcctgacct
gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc
1560aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc
cgacggctcc 1620ttcttcctct acagcaggct aaccgtggac aagagcaggt
ggcaggaggg gaatgtcttc 1680tcatgctccg tgatgcatga ggctctgcac
aaccactaca cacagaagag cctctccctg 1740tctctgggta aatga
1755175PRTArtificial SequenceSynthetic Construct 17Ser Phe Gly Met
His1 51817PRTArtificial SequenceSynthetic Construct 18Tyr Ile Asn
Gly Gly Ser Ser Thr Ile Phe Tyr Ala Asn Ala Val Lys1 5 10
15Gly1911PRTArtificial SequenceSynthetic Construct 19Tyr Ala Ser
Tyr Gly Gly Gly Ala Met Asp Tyr1 5 102016PRTArtificial
SequenceSynthetic Construct 20Arg Ser Ser Gln Ser Ile Val His Asn
Asp Gly Asn Thr Tyr Phe Glu1 5 10 15217PRTArtificial
SequenceSynthetic Construct 21Lys Val Ser Asn Arg Phe Ser1
5229PRTArtificial SequenceSynthetic Construct 22Phe Gln Gly Ser Tyr
Val Pro Leu Thr1 523120PRTArtificial SequenceSynthetic Construct
23Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe Tyr Ala
Asn Ala Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Ala Ser Tyr Gly Gly Gly
Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 12024112PRTArtificial SequenceSynthetic Construct 24Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Asn 20 25
30Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala
35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu
Thr Ile65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Phe Gln Gly 85 90 95Ser Tyr Val Pro Leu Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100
105 110255PRTArtificial SequenceSynthetic Construct 25Gly Gly Gly
Gly Ser1 5268PRTArtificial SequenceSynthetic Construct 26Gly Gly
Gly Gly Ser Ala Ala Ala1 5275PRTArtificial SequenceSynthetic
Construct 27Ala Ser Thr Gly Ser1 52810PRTArtificial
SequenceSynthetic Construct 28Ala Ser Thr Gly Ser Gly Gly Gly Gly
Ser1 5 1029120PRTArtificial SequenceSynthetic Construct 29Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val
35 40 45Ala Tyr Ile Asn Gly Gly Ser Ser Thr Ile Phe Tyr Ala Asn Ala
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Ala Ser Tyr Gly Gly Gly Ala Met
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12030112PRTArtificial SequenceSynthetic Construct 30Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ser Ser Gln Ser Ile Val His Asn 20 25 30Asp Gly
Asn Thr Tyr Phe Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile65
70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln
Gly 85 90 95Ser Tyr Val Pro Leu Thr Phe Gly Cys Gly Thr Lys Val Glu
Ile Lys 100 105 11031412PRTHomo Sapiens 31Met Pro Leu Gln Leu Leu
Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn1 5 10 15Ser Leu Gln Leu Trp
Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu 20 25 30Gly Pro Leu Leu
Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr 35 40 45Leu Asp Tyr
Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg 50 55 60Asn Ser
Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser65 70 75
80Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly
85 90 95Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn
Leu 100 105 110Ser Thr Asp Ser Ala Ala Met Glu Ile Gln Thr Thr Gln
Pro Ala Ala 115 120 125Thr Glu Ala Gln Thr Thr Gln Pro Val Pro Thr
Glu Ala Gln Thr Thr 130 135 140Pro Leu Ala Ala Thr Glu Ala Gln Thr
Thr Arg Leu Thr Ala Thr Glu145 150 155 160Ala Gln Thr Thr Pro Leu
Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro 165 170 175Ala Ala Thr Glu
Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln 180 185 190Thr Thr
Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala 195 200
205Met Glu Ala Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr Thr
210 215 220Gln Thr Thr Ala Met Glu Ala Gln Thr Thr Ala Pro Glu Ala
Thr Glu225 230 235 240Ala Gln Thr Thr Gln Pro Thr Ala Thr Glu Ala
Gln Thr Thr Pro Leu 245 250 255Ala Ala Met Glu Ala Leu Ser Thr Glu
Pro Ser Ala Thr Glu Ala Leu 260 265 270Ser Met Glu Pro Thr Thr Lys
Arg Gly Leu Phe Ile Pro Phe Ser Val 275 280 285Ser Ser Val Thr His
Lys Gly Ile Pro Met Ala Ala Ser Asn Leu Ser 290 295 300Val Asn Tyr
Pro Val Gly Ala Pro Asp His Ile Ser Val Lys Gln Cys305 310 315
320Leu Leu Ala Ile Leu Ile Leu Ala Leu Val Ala Thr Ile Phe Phe Val
325 330 335Cys Thr Val Val Leu Ala Val Arg Leu Ser Arg Lys Gly His
Met Tyr 340 345 350Pro Val Arg Asn Tyr Ser Pro Thr Glu Met Val Cys
Ile Ser Ser Leu 355 360 365Leu Pro Asp Gly Gly Glu Gly Pro Ser Ala
Thr Ala Asn Gly Gly Leu 370 375 380Ser Lys Ala Lys Ser Pro Gly Leu
Thr Pro Glu Pro Arg Glu Asp Arg385 390 395 400Glu Gly Asp Asp Leu
Thr Leu His Ser Phe Leu Pro 405 41032402PRTHomo Sapiens 32Met Pro
Leu Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn1 5 10 15Ser
Leu Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu 20 25
30Gly Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr
35 40 45Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu
Arg 50 55 60Asn Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro
Glu Ser65 70 75 80Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly
Leu Asp Ala Gly 85 90 95Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala
Asn Met Gly Asn Leu 100 105 110Ser Thr Asp Ser Ala Ala Met Glu Ile
Gln Thr Thr Gln Pro Ala Ala 115 120 125Thr Glu Ala Gln Thr Thr Pro
Leu Ala Ala Thr Glu Ala Gln Thr Thr 130 135 140Arg Leu Thr Ala Thr
Glu Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu145 150 155 160Ala Gln
Thr Thr Pro Pro Ala Ala Thr Glu Ala Gln Thr Thr Gln Pro 165 170
175Thr Gly Leu Glu Ala Gln Thr Thr Ala Pro Ala Ala Met Glu Ala Gln
180 185 190Thr Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Pro Pro
Ala Ala 195 200 205Met Glu Ala Gln Thr Thr Gln Thr Thr Ala Met Glu
Ala Gln Thr Thr 210 215 220Ala Pro Glu Ala Thr Glu Ala Gln Thr Thr
Gln Pro Thr Ala Thr Glu225 230 235 240Ala Gln Thr Thr Pro Leu Ala
Ala Met Glu Ala Leu Ser Thr Glu Pro 245 250 255Ser Ala Thr Glu Ala
Leu Ser Met Glu Pro Thr Thr Lys Arg Gly Leu 260 265 270Phe Ile Pro
Phe Ser Val Ser Ser Val Thr His Lys Gly Ile Pro Met 275 280 285Ala
Ala Ser Asn Leu Ser Val Asn Tyr Pro Val Gly Ala Pro Asp His 290 295
300Ile Ser Val Lys Gln Cys Leu Leu Ala Ile Leu Ile Leu Ala Leu
Val305 310 315 320Ala Thr Ile Phe Phe Val Cys Thr Val Val Leu Ala
Val Arg Leu Ser 325 330 335Arg Lys Gly His Met Tyr Pro Val Arg Asn
Tyr Ser Pro Thr Glu Met 340 345 350Val Cys Ile Ser Ser Leu Leu Pro
Asp Gly Gly Glu Gly Pro Ser Ala 355 360 365Thr Ala Asn Gly Gly Leu
Ser Lys Ala Lys Ser Pro Gly Leu Thr Pro 370 375 380Glu Pro Arg Glu
Asp Arg Glu Gly Asp Asp Leu Thr Leu His Ser Phe385 390 395 400Leu
Pro3325PRTArtificial SequenceSynthetic Construct 33Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly
Ser Gly Gly Gly Gly Ser 20 253410PRTArtificial SequenceSynthetic
Construct 34Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
103511PRTArtificial SequenceSynthetic Construct 35Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly1 5 103612PRTArtificial
SequenceSynthetic Construct 36Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly1 5 10
* * * * *