U.S. patent application number 17/557898 was filed with the patent office on 2022-06-16 for engineered antibody for inhibition of fibrosis.
The applicant listed for this patent is Thomas Jefferson University. Invention is credited to Andrzej Fertala, Andrzej Steplewski.
Application Number | 20220185873 17/557898 |
Document ID | / |
Family ID | 1000006167899 |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220185873 |
Kind Code |
A1 |
Fertala; Andrzej ; et
al. |
June 16, 2022 |
ENGINEERED ANTIBODY FOR INHIBITION OF FIBROSIS
Abstract
A chimeric, humanized or single-chain antibody contains a light
chain variable region containing the complementarity determining
regions of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and a heavy
chain variable region containing the complementarity determining
regions SEQ ID NO:5 and SEQ ID NO:6. The antibody or antibody
fragment thereof is capable of binding the C-terminal telopeptide
of the .alpha.2(I) chain of human collagen I, and is useful in the
treatment of diseases or disorders associated with excessive
collagen fibril.
Inventors: |
Fertala; Andrzej; (Voorhees,
NJ) ; Steplewski; Andrzej; (Phoenixville,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Thomas Jefferson University |
Philadelphia |
PA |
US |
|
|
Family ID: |
1000006167899 |
Appl. No.: |
17/557898 |
Filed: |
December 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16705538 |
Dec 6, 2019 |
11208472 |
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17557898 |
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15699083 |
Sep 8, 2017 |
10501533 |
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16705538 |
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14391585 |
Oct 9, 2014 |
9777055 |
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PCT/US2013/027787 |
Feb 26, 2013 |
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15699083 |
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61636073 |
Apr 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/565 20130101; C07K 2317/24 20130101; C07K 2317/76
20130101; C07K 2317/622 20130101; A61K 2039/505 20130101; C07K
2317/92 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1. A chimeric, humanized or single-chain antibody which comprises a
light chain variable region comprising complementarity determining
regions comprising the amino acid sequences SEQ ID NO: 1, SEQ ID
NO:2 and SEQ ID NO:3, and a heavy chain variable region comprising
complementarity determining regions comprising the amino acid
sequences SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, or a fragment
of said chimeric or humanized antibody, which antibody or antibody
fragment binds the C-terminal telopeptide of the a2(I) chain of
human collagen I.
2. The antibody or antibody fragment according to claim 1, which is
a chimeric antibody or fragment of a chimeric antibody, which
fragment binds the C-terminal telopeptide of the a2(I) chain of
human collagen.
3. The chimeric antibody according to claim 2, which comprises an
antibody light chain comprising a light variable region having the
amino acid sequence shown in SEQ ID NO.T0, and an antibody heavy
chain comprising a heavy chain variable region having the amino
acid sequence SEQ ID NO: l 1, or a fragment of said chimeric
antibody which binds the C-terminal telopeptide of the a2(I) chain
of human collagen.
4. The chimeric antibody according to claim 3, wherein said
antibody light chain comprises a human antibody light chain
constant region, and said antibody heavy chain comprises a human
antibody heavy chain constant region, or a fragment of said
chimeric antibody which binds the C-terminal telopeptide of the
oc2(I) chain of human collagen.
5. The chimeric antibody according to claim 4, wherein the human
constant regions comprise IgG constant regions.
6. The antibody or antibody fragment according to claim 1, which is
a humanized antibody or fragment of a humanized antibody, which
fragment binds the C-terminal telopeptide of the ct2(I) chain of
human collagen.
7. The humanized antibody according to claim 6, which comprises a
human antibody framework region and/or a human antibody constant
region, or a fragment of said humanized antibody which binds the
C-terminal telopeptide of the 2(I) chain of human collagen.
8. The humanized antibody according to claim 6, which comprises a
human antibody constant region.
9. The humanized antibody according to claim 8, wherein the
constant region comprises an IgG constant region.
10. The antibody according to claim 1, which is a single chain
antibody.
11. The single chain antibody according to claim 10, wherein the
single chain antibody comprises a light chain variable region
having the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO: 10
or SEQ ID NO:26, and a heavy chain variable region having the amino
acid sequence shown in SEQ ID NO: l 1.
12. The single chain antibody according to claim 11, wherein the
antibody further comprises a linker comprising a sequence of one or
more amino acids linking said light chain variable region and said
heavy chain variable region.
13. The single chain antibody according to claim 12, wherein the
linker comprises the amino acid sequence Gly-Gly-Ser or the amino
acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).
14. The single chain antibody according to claim 12, wherein the
linker connects the carboxy terminus of said light chain variable
region to the amino terminus of said heavy chain variable
region.
15. The single chain antibody according to claim 13, wherein the
linker comprises from two to twelve repeats of the amino acid
sequence Gly-Gly-Ser, or from two to twelve repeats of the amino
acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).
16. The single chain antibody according to claim 15, comprising the
amino acid sequence shown in SEQ ID NO: 12.
17. The single chain antibody according to claim 12, wherein the
linker connects the carboxy terminus of said heavy chain variable
region to the amino terminus of said light chain variable
region.
18. The single chain antibody according to claim 17, wherein the
linker comprises from two to twelve repeats of the amino acid
sequence Gly-Gly-Ser, or from two to twelve repeats of the amino
acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).
19. The single chain antibody according to claim 18, comprising the
amino acid sequence shown in SEQ ID NO: 16.
20. A polynucleotide for expressing a chimeric antibody light
chain, said polynucleotide comprising a first segment encoding an
antibody light chain variable region having the amino acid sequence
shown in SEQ ID NO: 10 and a second segment encoding a human
antibody light chain constant region.
21. The polynucleotide according to claim 20, wherein the first
segment comprises the nucleotide sequence shown in SEQ ID
NO:22.
22. A polynucleotide for expressing a chimeric antibody heavy
chain, said polynucleotide comprising a first segment encoding an
antibody heavy chain variable region having the amino acid sequence
shown in SEQ ID NO: l 1 and a second segment encoding a human
antibody heavy chain constant region.
23. The polynucleotide according to claim 22, wherein the first
segment comprises the nucleotide sequence shown in SEQ ID
NO:23.
24. A polynucleotide for expressing a single chain antibody
comprising a first segment encoding an antibody light chain
variable region having the amino acid sequence shown in SEQ ID
NO:9, SEQ ID NO: 10 or SEQ ID NO:26, and a second segment encoding
an antibody heavy chain variable region comprising the amino acid
sequence shown in SEQ ID NO: 11.
25. The polynucleotide of claim 24 comprising a linker segment
between said first and second segments, said linker segment
encoding a linker comprising one or more amino acids.
26. The polynucleotide according to claim 25, wherein the linker
segment encodes a linker comprising from two to twelve repeats of
the amino acid sequence Gly-Gly-Ser, or from two to twelve repeats
of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).
27. The polynucleotide according to claim 26 wherein the first DNA
segment comprises the nucleotide sequence SEQ ID NO:22, SEQ ID
NO:27 or SEQ ID NO:28, and the second DNA segment comprises the
nucleotide sequence SEQ ID NO:23.
28. The polynucleotide according to claim 24, encoding a single
chain antibody comprising the amino acid sequence SEQ ID NO:
12.
29. The polynucleotide according to claim 28, comprising the
nucleotide sequence SEQ ID NO: 13.
30. The polynucleotide according to claim 24, encoding a single
chain antibody comprising the amino acid sequence SEQ ID NO:
16.
31. The polynucleotide according to claim 30, comprising the
nucleotide sequence SEQ ID NO: 17.
32. A pharmaceutical composition comprising an antibody or antibody
fragment according to claim 1 and a pharmaceutically acceptable
carrier.
33. A method of treating a disease or disorder associated with
excessive collagen fibril formation in a subject, comprising
administering to the subject an effective amount of an antibody or
antibody fragment according to claim 1.
34. The method according to claim 33, wherein the disease or
disorder comprises scar formation.
35. The method according to claim 34, wherein the disease or
disorder comprises fibrosis.
36. The method according to claim 35, wherein the fibrosis
comprises pulmonary fibrosis, idiopathic pulmonary fibrosis,
cirrhosis, endomyocardial fibrosis, mediastinal fibrosis,
myelofibrosis, retroperitoneal fibrosis, progressive massive
fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid
formation, myocardial infarction, scleroderma/systemic sclerosis,
arthrofibrosis, or adhesive capsulitis.
37. The method according to claim 35 wherein the fibrosis results
from a surgical procedure.
38. The method according to claim 37 wherein the surgery is
abdominal surgery, plastic surgery, glaucoma surgery or surgery for
implantation of a medical implant or device.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/705,538, filed Dec. 6, 2019, now allowed,
which is a divisional of U.S. patent application Ser. No.
15/699,083, filed Sep. 8, 2017 under 35 U.S.C. .sctn. 120, now U.S.
Pat. No. 10,501,533 issued Dec. 10, 2019, which is a divisional of
U.S. patent application Ser. No. 14/391,585, filed Oct. 9, 2014,
now U.S. Pat. No. 9,777,055 issued Oct. 3, 2017, which is a 35
U.S.C. .sctn. 371 National Stage Entry of International Patent
Application No. PCT/US13/27787, filed Feb. 26, 2013, which claims
the benefit of U.S. Provisional Patent Application No. 61/636,073,
filed Apr. 20, 2012. The entire disclosures of the aforesaid
applications are incorporated herein by reference in their
entirety.
REFERENCE TO GOVERNMENT GRANT
[0002] The invention was made with government support under grant
5RO1 AR048544-05 and IR21AR06118-01 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 14, 2021, is named 205961_7033US4_Sequence Listing.txt and
is 30 KB in size.
FIELD OF THE INVENTION
[0004] The invention relates to engineered antibodies for the
treatment of fibrotic disorders.
BACKGROUND OF THE INVENTION
[0005] Collagen I, the most abundant structural protein of
connective tissue such as skin, bone and tendon, is first
synthesized as a precursor molecule, procollagen. Formation of
collagen fibrils is initiated by enzymatic processing of
procollagen to expose telopeptides, which engage in site-specific
intermolecular interactions to drive collage self assembly. In
vivo, collagen fibrils are stabilized by the covalent cross-links
formed between fibril- incorporated collagen molecules. Self
assembly of collagen molecules results in collagen fibrils, the
main component of fibrotic lesions, particularly scarring.
[0006] Collagen/collagen binding is mediated through the
interaction of the C-terminal .alpha.1(I) and .alpha.2(I)
telopeptides of one collagen molecule, and the Triple-helical
Telopeptide-Binding Region (T-TBR) of another binding partner. The
T-TBR is located within an .alpha.1(I) chain in the region flanked
by resides 776 and 796 (Prockop, et al. (1998) J Biol Chem. 273,
15598-15604).
[0007] Fibrosis is the formation of excess fibrous connective
tissue in an organ or tissue in a reparative or reactive process.
Types of fibrosis include, for example, pulmonary fibrosis (lungs),
idiopathic pulmonary fibrosis (where the cause is unknown),
cirrhosis (liver), endomyocardial fibrosis (heart), mediastinal
fibrosis (soft tissue of the mediastinum), myelofibrosis (bone
marrow), retroperitoneal fibrosis (soft tissue of the
retroperitoneum), progressive massive fibrosis (lungs), nephrogenic
systemic fibrosis (skin), Crohn's Disease intestine, keloid (skin),
myocardial infarction (heart), scleroderma/systemic sclerosis
(skin, lungs), arthrofibrosis (knee, shoulder, other joints) and
some forms of adhesive capsulitis (shoulder).
[0008] Although the fibrotic changes seen in excessive scarring may
be triggered in many ways, such as trauma, accidental injury or
surgical procedures, most of them are developed through
fundamentally similar pathways that, in the end, lead to altering a
number of functions of involved tissues. For instance, after
surgery in the abdomen, the formation of excessive scar tissue
around abdominal organs often interferes with their functionality.
After plastic surgery to the face, the formation of excessive scar
tissue frequently compromises the benefits of the surgery.
Excessive scar formation also presents a major complication in the
eye after glaucoma surgery performed to maintain a lamellar channel
from the subconjunctival space to the anterior chamber. Frequently,
however, the excessive scar formation closes this pressure-reducing
channel, thereby forcing the intraocular pressure to rise (Addicks,
et al. (1983) Arch Ophthalmol. 101, 795-798). Yet another
significant problem with excessive formation of fibrous deposits is
the foreign body response to medical devices and materials
implanted in the human body (Anderson, et al. (2008) Semin Immunol.
20, 86-100). Moreover, excessive scarring of the vocal folds may
severely alter their ability to vibrate, thereby causing a number
of voice disorders (Lim, et al. (2006) Ann Otol Rhinol Laryngol.
115, 921-929). Another medical problem of localized fibrosis is the
formation of keloids, excessive scars for which there are no
successful treatment methods. This particular scarring is an
ongoing and rising problem; as keloids are more common in Americans
of African and Asian descent, it is expected that in the near
future the number of keloid cases in the USA will increase due to
the foreseen rise in the percentage of these ethnic groups (Taylor,
et al. (2002) J Am Acad Dermatol. 46, S41-62).
[0009] To date, no effective therapeutics for excessive fibrosis
are available. What is needed are therapeutics and therapeutic
methods that can prevent the excessive deposition of collagen
fibrils that is characteristic of fibrotic processes, and to reduce
localized and systemic fibrotic lesions.
SUMMARY OF THE INVENTION
[0010] A chimeric, humanized or single-chain antibody is provided.
The antibody comprises a light chain variable region comprising
complementarity determining regions comprising the amino acid
sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and a heavy
chain variable region comprising the complementarity determining
regions comprising the amino acid sequences SEQ ID NO:5 and SEQ ID
NO:6. Also provided are fragments of said chimeric or humanized
antibodies. The antibody or antibody fragment is capable of binding
the C-terminal telopeptide of the .alpha.2(I) chain of human
collagen I.
[0011] In one embodiment, the antibody or antibody fragment is a
chimeric antibody or chimeric antibody fragment. In one embodiment,
the chimeric antibody comprises an antibody light chain comprising
a light variable region having the amino acid sequence SEQ ID
NO:10, and an antibody heavy chain comprising a heavy chain
variable region having the amino acid sequence SEQ ID NO:11. In
another embodiment, a fragment of said chimeric antibody is
provided, which antibody fragment binds the C-terminal telopeptide
of the .alpha.2(I) chain of human collagen.
[0012] In embodiments of the chimeric antibody, the antibody light
chain comprises a human antibody light chain constant region, and
the antibody heavy chain comprises a human antibody heavy chain
constant region. In another embodiment, a fragment of said chimeric
antibody is provided, which antibody fragment binds the C-terminal
telopeptide of the .alpha.2(I) chain of human collagen. In some
embodiments, the constant region comprises an IgG constant
region.
[0013] In another embodiment, the antibody or antibody fragment is
a humanized antibody or fragment of a humanized antibody, which
fragment binds the C-terminal telopeptide of the .alpha.2(I) chain
of human collagen. In some embodiments, the humanized antibody or
antibody fragment comprises a human antibody framework region
and/or a human antibody constant region. In some embodiment, the
constant region comprises an IgG constant region. Also provided are
fragment of said humanized antibody which bind the C-terminal
telopeptide of the .alpha.2(I) chain of human collagen.
[0014] In other embodiments, the antibody is a single chain
antibody. In one embodiment, the single chain antibody comprises a
light chain variable region having the amino acid sequence shown in
SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:26, and a heavy chain
variable region having the amino acid sequence shown in SEQ ID
NO:11.
[0015] In certain embodiments, the single chain antibody comprises
a linker comprising a sequence of amino acids that links the light
chain variable region and the heavy chain variable region.
[0016] In some embodiments, the linker connects the carboxy
terminus of the light chain variable region to the amino terminus
of the heavy chain variable region. In other embodiments, the
linker connects the carboxy terminus of the heavy chain variable
region to the amino terminus of the light chain variable
region.
[0017] In some embodiments, the linker may comprise the amino acid
sequence Gly-Gly-Ser, or the amino acid sequence
Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29). In some embodiments, the linker
comprises from two to twelve repeats of the amino acid sequence
Gly-Gly-Ser or from two to twelve repeats of the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29). In some embodiments,
the aforesaid linker connects the carboxy terminus of the light
chain variable region to the amino terminus of the heavy chain
variable region. In other embodiments, the aforesaid linker
connects the carboxy terminus of the heavy chain variable region to
the amino terminus of the light chain variable region.
[0018] In an embodiment, the single chain antibody comprises the
amino acid sequence shown in SEQ ID NO:12. In another embodiment,
the single chain antibody comprises the amino acid sequence shown
in SEQ ID NO:16.
[0019] Provided is a polynucleotide for expressing a chimeric
antibody light chain, said polynucleotide comprising a first
segment encoding an antibody light chain variable region having the
amino acid sequence SEQ ID NO:10 and a second segment encoding a
human antibody light chain constant region. In one embodiment, of
the aforesaid polynucleotide, the first segment comprises the
nucleotide sequence SEQ ID NO:22.
[0020] Provided is a polynucleotide for expressing a chimeric
antibody heavy chain, said polynucleotide comprising a first
segment encoding an antibody heavy chain variable region having the
amino acid sequence SEQ ID NO:11 and a second segment encoding a
human antibody heavy chain constant region. In one embodiment of
the aforesaid polynucleotide, the first segment comprises the
nucleotide sequence SEQ ID NO:23.
[0021] Provided is a polynucleotide for expressing a single chain
antibody comprising a first segment encoding an antibody light
chain variable region having the amino acid sequence shown in SEQ
ID NO:9, SEQ ID NO:10 or SEQ ID NO:26, and a second segment
encoding an antibody heavy chain variable region comprising the
amino acid sequence shown in SEQ ID NO:11. In certain embodiments,
the polynucleotide comprises a linker segment between said first
and second segments. The linker segment encodes a linker comprising
one or more amino acids. In certain embodiments, the linker segment
encodes a linker comprising from two to twelve repeats of the amino
acid sequence Gly-Gly-Ser, or from two to twelve repeats of the
amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29). In
embodiments, the first DNA segment comprises the nucleotide
sequence SEQ ID NO:22, SEQ ID NO:27 or SEQ ID NO:28, and the second
DNA segment comprises the nucleotide sequence SEQ ID NO:23.
[0022] In an embodiment, the polynucleotide for expressing a single
chain antibody encodes a single chain antibody comprising the amino
acid sequence SEQ ID NO:12. In an embodiment, the aforesaid
polynucleotide comprises the nucleotides sequence SEQ ID NO:13.
[0023] In an embodiment, the polynucleotide for expressing a single
chain antibody encodes a single chain antibody comprising the amino
acid sequence SEQ ID NO:16. In an embodiment, the aforesaid
polynucleotide comprises the nucleotide sequence SEQ ID NO:17.
[0024] Also provided are pharmaceutical compositions comprising the
aforementioned chimeric, humanized or single-chain antibody, or
fragment of a chimeric or humanized antibody, and a
pharmaceutically acceptable carrier.
[0025] Also provided is a method of treating a disease or disorder
associated with excessive collagen fibril formation in a subject,
comprising administering to the subject an effective amount of the
aforesaid antibody or antibody fragment. In one embodiment, the
disease or disorder comprises scar formation. In some embodiments,
the disease or disorder comprises fibrosis. Non-limiting examples
of such fibroses include: pulmonary fibrosis, idiopathic pulmonary
fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis,
myelofibrosis, retroperitoneal fibrosis, progressive massive
fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid
formation, myocardial infarction, scleroderma/systemic sclerosis,
arthrofibrosis, and adhesive capsulitis.
[0026] In certain embodiments of the method of treatment, the
fibrosis results from a surgical procedure, e.g., abdominal
surgery, plastic surgery, glaucoma surgery or surgery for
implantation of a medical implant or device.
[0027] In another embodiment, the aforesaid chimeric, humanized or
single-chain antibody, or fragment of a chimeric or humanized
antibody is provided for use in medicine. In another embodiment,
the aforesaid chimeric, humanized or single-chain antibody, or
fragment of a chimeric or humanized antibody is for treating a
disease or disorder associated with excessive collagen fibril
formation in a subject. In another embodiment, the aforesaid
chimeric, humanized or single-chain antibody, or fragment of a
chimeric or humanized antibody is used for the preparation of a
medicament for treating a disease or disorder associated with
excessive collagen fibril formation in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 shows the amino acid sequence (SEQ ID NO:7) of the
light chain variable region of an engineered antibody of the
invention. The complementarity determining regions (CDRs) are
double-underlined. The first fifteen amino acids in FIG. 1 comprise
a signal sequence. The signal sequenced is single-underlined.
[0029] FIG. 2 shows the amino acid sequence (SEQ ID NO:8) of the
heavy chain variable region of an engineered antibody of the
invention. The first nineteen amino acids in FIG. 2 comprise a
signal sequence. The signal sequenced is single-underlined.
[0030] FIG. 3 shows the DNA (SEQ ID NO:15) and amino acid (SEQ ID
NO:14) sequences of an scFv for bacterial expression. The sequences
of the V.sub.H and V.sub.L regions as well as that of the linker
are indicated. The black boxes indicate insertions made during the
cloning process. A FLAG-tag is also indicated.
[0031] FIG. 4 is a schematic of the scFv construct of FIG. 3 cloned
into the pET-22 vector.
[0032] FIG. 5A shows an electrophoretic assay of a refolded scFv of
the invention expressed in bacteria purified with anti-FLAG
antibody. Electrophoretic migration of the purified single chain
antibody indicates the predicted mass.
[0033] FIG. 5B shows an electrophoretic assay of the refolded
single chain antibody expressed in bacteria and purified by ion
exchange chromatography. The fraction including the scFv was eluted
with 200 mM NaCl, while contaminating proteins were eluted with 500
mM NaCl.
[0034] FIG. 6 shows a chromatography profile of the same scFv
analyzed by HPLC size exclusion. The three main peaks indicate the
single chain antibody monomers and oligomers.
[0035] FIG. 7 shows the results of a Western blot analysis of the
binding of the same scFv to collagen .alpha.2-chain. The lines in
FIG. 7 depict different repeats of the assay, and were identified
as procollagen I .alpha.2 chains, thereby indicating the binding
specificity of the single chain antibody for collagen
.alpha.2-chain.
[0036] FIG. 8 shows the inhibition of collagen fibril deposition in
cultures of keloid-derived human fibroblasts by the scFv: (I) non
treated, (II) treated with tissue growth factor .beta.(TGF.beta.)
and single chain antibody, and (III) treated only with TGF-.beta..
Cell layers consisting of collagen-rich extracellular matrix and
cells were collected and analyzed for the presence of collagen I
(Col-I), a main constituent of collagen fibrils. An equal number of
cells in the analyzed groups is indicated by comparable amounts of
GAPDH. The analyzed groups and the collagen I marker (Cm) are
indicated.
[0037] FIGS. 9A-9C show the DNA coding sequence (upper sequence,
which is SEQ ID NO:19), the DNA non-coding sequence (middle
sequence, which is SEQ ID NO:30), and amino acid (SEQ ID NO:18)
sequences of the construct designated A_L_K, for the expression of
an scFv in yeast. Restriction sites utilized in the cloning
strategy are indicated.
[0038] FIG. 10 is a schematic of the scFv construct of FIGS. 9A-9C
cloned into the pPIC-9K plasmid. Restriction sites utilized in the
cloning strategy are indicated.
[0039] FIG. 11 shows the DNA (SEQ ID NO:20) and amino acid (SEQ ID
NO:8) sequences of a construct encoding a mouse heavy chain
variable region (mV.sub.H), for the preparation of a further
construct for expressing a chimeric antibody heavy chain consisting
of the mV.sub.H and the constant region of a human .gamma. chain
(mV.sub.H-h.gamma.). Restriction sites utilized in the cloning
strategy are indicated.
[0040] FIG. 12 is a schematic of a plasmid for expression of the
mV.sub.H-h.gamma., utilizing the mV.sub.H construct of FIG. 11. The
mV.sub.H construct was cloned into the pFUSE-CHIg-hG1 plasmid as
shown in FIG. 12 to express the mV.sub.H-h.gamma.. Restriction
sites utilized in the cloning strategy are indicated.
[0041] FIG. 13 shows the DNA (SEQ ID NO:21) and amino acid (SEQ ID
NO:7) sequences of a construct encoding a mouse light chain
variable region (mV.sub.L), for the preparation of a plasmid for
expressing a chimeric antibody heavy chain consisting of the
mV.sub.L and the constant region of a human .kappa. chain
(mV.sub.H-h.kappa.). Restriction sites utilized in the cloning
strategy are indicated.
[0042] FIG. 14 is a schematic of a plasmid for expression of the
chimeric mV.sub.L-h.kappa., utilizing the mV.sub.L construct of
FIG. 13. The mV.sub.L construct was cloned into the pFUSE2-CLIg-hk
plasmid as shown in FIG. 14 to express the mV.sub.H-h.kappa..
Restriction sites utilized in the cloning strategy are
indicated.
[0043] FIG. 15A is a Western blot analysis under reducing
conditions showing that specific antibodies against IgG .gamma. and
.kappa. chains (Anti-.gamma.; Anti-.kappa.) reacted with a chimeric
antibody of the invention secreted by CHO cells. The results
confirm the presence of .gamma. and .kappa. chains in the chimeric
antibody (chimeric-.gamma.; chimeric-.kappa.). Human IgG containing
a .kappa. chain was used as positive marker (h-IgG(.kappa.)). The
chimeric antibody was composed of a murine heavy .alpha. chain
variable region (SEQ ID NO:8)/human .gamma. constant region chain
and a murine light chain .kappa. region (SEQ ID NO:7)/human .kappa.
constant region chain.
[0044] FIG. 15B is a Western blot analysis of the same chimeric
antibody under non-reducing conditions with anti-human .gamma. and
anti-human .kappa. antibodies. The results indicate that the
antibody heavy and light chains (the component mV.sub.H-h.gamma.
and mV.sub.L-h.kappa. chains) were covalently linked via disulfide
bonds, thereby indicating the formation of the chimeric antibody.
The presence of a high molecular band (CHO-IgG) indicates
production of chimeric IgG molecules by the CHO cells. Human IgG
with the .kappa. chain was used as positive marker
(h-IgG(.kappa.)).
[0045] FIG. 16 is a Western blot assay showing the binding of the
purified chimeric antibody to its target, .alpha.2Ct, as detected
by chemiluminescence with the use of the anti-human .gamma.
antibodies conjugated with horseradish peroxidase (HRP). Positive
bands were identified as those corresponding to intact procollagen
I .alpha.2 chain (upper band) and its partially degraded form.
Native human IgG with the .kappa. chain was used as a positive
marker (Bethyl Laboratories Inc.).
[0046] FIG. 17 shows association and dissociation curves
illustrating kinetics of the binding between procollagen and the
following anti-.alpha.2Ct antibody variants: the original
anti-.alpha.2Ct mouse IgA antibody (IgA) from which the mV.sub.H
and mV.sub.L were obtained; the bacterial-derived scFv variant
(scFv) of FIG. 3; the mouse-human chimeric IgG (chimeric IgG); and
human IgG with the light .kappa. chain (human-IgG) (control). In
each panel, the curves represent association and dissociation
events during the interaction between immobilized procollagen I and
free antibody variants present at concentrations ranging from
4.times.10.sup.-7 M to 1.25.times.10.sup.-8 M. For each assay, the
association rate constants (k.sub.on) and the dissociation rate
constants (k.sub.off) were obtained, and the equilibrium
dissociation constants (K.sub.D) values were calculated from a
ratio of k.sub.off/k.sub.on.
[0047] FIG. 18A is a Western blot assay of scFv variant prepared in
yeast from the A_L_K construct. The lanes show the secretion of
various yeast clones cultured in His(-) conditions. Cell culture
media from non-transformed yeast cells (left lane, "Ctrl") are
negative for the scFv variant.
[0048] FIG. 18B shows the result of PCR assays of yeast clones
producing scFv variants from the A_L_K DNA construct, and from
another DNA construct, K_L_A. The presence of the DNA constructs in
the genomes of the analyzed clones is indicated.
[0049] FIG. 19 shows an electrophoretic assay of the A_L_K yeast
scFv variant after purification by nickel column. Electrophoretic
migration of the purified scFv in denaturing/reducing conditions
indicates its predicted mass.
[0050] FIG. 20 is a Western blot analysis showing in lane (A)
electrophoresis of the purified human procollagen I in a
polyacrylamide gel, showing staining of procollagen I .alpha.1 and
.alpha.2 chains. The procollagen chains were transferred to a
nitrocellulose membrane and probed with His-tagged yeast-derived
A_L_K scFv variant. The A_L_K scFv variant bound to its specific
target procollagen I .alpha.2 chain, as shown in lane (B).
DEFINITIONS
[0051] As used herein, each of the following terms has the meaning
associated with it in this section.
[0052] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0053] The term "about" will be understood by persons of ordinary
skill in the art and will vary to some extent depending on the
context in which it is used. As used herein, "about" is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably
.+-.0.1%.
[0054] Unless otherwise specified herein, the terms "antibody" and
"antibodies" broadly encompass naturally-occurring forms of
antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies as
well as derivatives have at least an antigenic binding site.
Antibody derivatives may comprise a protein or chemical moiety
conjugated to an antibody.
[0055] By "chimeric antibody" is meant an antibody molecule in
which different portions are derived from different animal species,
such as those having a variable region derived from a murine
antibody and a human immunoglobulin constant region. Typically, a
chimeric antibody is composed of a variable region comprising light
and heavy chain variable regions, and a constant region derived or
obtained from a human antibody.
[0056] A "humanized antibody" refers to an antibody in which the
complementarity defining regions (CDRs) of an antibody of a
non-human mammal, e.g., mouse, are grafted to a human antibody. The
variable domain of each of an antibody heavy chain and light chain
comprise three CDRs; the intervening sequence segments are
"framework segments". Each variable domain is composed of four
framework segments. In a humanized antibody, the framework segments
are typically of human origin.
[0057] A "single chain antibody", also known as a "single-chain
variable fragment" (scFv) is a fusion protein of the variable
regions of the heavy and light chains of an immunoglobulin, wherein
the regions are optionally connected by a linker. As used herein,
"single chain antibody" or "single-chain variable fragment"
includes such fusion proteins, and also multimers (linear or
branched) formed of such fusion proteins.
[0058] A "polynucleotide" as utilized in the practice of the
present invention may include any polymer or oligomer of pyrimidine
and purine bases, preferably cytosine, thymine, and uracil, and
adenine and guanine, respectively. The polynucleotide may comprise
any deoxyribonucleotide, ribonucleotide or peptide nucleic acid
component, and any chemical variants thereof, such as methylated,
hydroxymethylated or glucosylated forms of these bases, and the
like. The polynucleotide may comprise DNA or RNA, or a mixture
thereof, and may exist permanently or transitionally in
single-stranded or double-stranded form, including homoduplex,
heteroduplex, and hybrid states.
[0059] As used herein, the term "subject" or "patient" refers to
any animal (e.g., a mammal), including, but not limited to humans,
non-human primates, rodents, and the like. Typically, the terms
"subject" and "patient" are used interchangeably herein in
reference to a human subject.
[0060] The term "treating" as used herein, unless otherwise
indicated, means reversing, alleviating, inhibiting the progress
of, or preventing, either partially or completely, an indicated
disease or disorder. The term "treatment" as used herein, unless
otherwise indicated, refers to the act of treating. The term
"treating" does not necessarily mean that the disease or disorder
will, in fact, be eliminated.
[0061] The term "therapeutically effective amount" or "effective
amount" means the amount of a subject compound or combination that
will elicit the biological or medical response of a tissue, system,
animal or human that is being sought.
[0062] As envisioned in the present invention with respect to the
disclosed compositions of matter and methods, in one aspect the
embodiments of the invention comprise the components and/or steps
disclosed herein. In another aspect, the embodiments of the
invention consist essentially of the components and/or steps
disclosed herein. In yet another aspect, the embodiments of the
invention consist of the components and/or steps disclosed
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0063] Engineered antibodies are provided that bind to the
C-terminal telopeptides of the .alpha.2-chain of collagen. The
engineered antibodies inhibit the formation of collagen fibrils.
Free collagen molecules that do not incorporate into fibrils are
readily accessible for degradation by enzymes present in the
extracellular space (Prockop and Fertala, J. Biol. Chem.
273:15598-15604 (1988); Chang et al., Diabetes 29:778-781 (1980)).
The engineered antibodies comprise a light chain variable region
comprising the three complementarity determining regions having the
amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3, and a heavy chain variable region comprising the three
complementarity determining regions having the amino acid sequences
shown in SEQ ID NO:5 and SEQ ID NO:6. The engineered antibodies are
capable of binding the C-terminal telopeptide of the .alpha.2(I)
chain of human collagen I.
[0064] In one embodiment, the engineered antibody is a chimeric
antibody comprising the mouse light chain variable region
(mV.sub.L) having the amino acid sequence of SEQ ID NO:10, and the
mouse heavy chain variable region (mV.sub.H) having the amino acid
sequence of SEQ ID NO:11. The mouse light chain variable region and
mouse heavy chain variable regions, with predicted signal peptides,
are shown in FIGS. 1 and 2, respectively. The mV.sub.L
complementarity determining regions are shown by double underlining
in FIG. 1. The first fifteen amino acids in FIG. 1 (indicated by
single underlining) comprise the signal sequence for the mV.sub.L.
The first nineteen amino acids in FIG. 2 (indicated by single
underlining) comprise the signal sequence for the mV.sub.H.
[0065] Using the sequences encoding the above light and heavy chain
variable regions, chimeric antibodies may be produced by any of the
well-known techniques for production of chimeric antibodies
(Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-5;
Neuberger et al., 1984, Nature, 312:604-8; Takeda et al., 1985,
Nature, 314:452-4). Accordingly, a DNA molecule encoding the light
chain variable region SEQ ID NO:10, e.g., the DNA molecule having
the nucleotide sequence of SEQ ID NO:22, is prepared. The DNA
molecule may further encode a signal sequence for the light chain
variable region, such as the fifteen amino acid signal sequence
shown in FIG. 1 by single underlining. SEQ ID NO:7 is the amino
acid sequence of the polypeptide comprising the signal sequence and
light chain variable region shown in FIG. 1. SEQ ID NO:7 may be
encoded by, for example, the nucleotide sequence of SEQ ID
NO:24.
[0066] A DNA molecule encoding the heavy chain variable region SEQ
ID NO:11, e.g., the DNA molecule having the nucleotide sequence of
SEQ ID NO:23, is prepared. The DNA molecule may further encode a
signal sequence for the heavy chain variable region, such as the
nineteen amino acid signal sequence shown in FIG. 2 by single
underlining. SEQ ID NO:8 is the amino acid sequence of the
polypeptide comprising the signal sequence and heavy chain variable
region shown in FIG. 2. SEQ ID NO:8 may be encoded by, for example,
the nucleotide sequence of SEQ ID NO:25.
[0067] The DNA molecules encoding the light and heavy chain
variable regions are then ligated into vector DNA and expressed
using, for example, any of the available ligation kits to construct
a recombinant vector. See e.g., J. Sambrook et, Molecular Cloning,
Cold Spring Harbor Laboratory Press, 1989.
[0068] In one embodiment, a chimeric antibody may be prepared by
ligating a mouse leader sequence and a variable region sequence
present in a cloned mouse cDNA to a sequence coding for a human
antibody constant region already present in an expression vector of
a mammalian cell. Alternatively, a mouse leader sequence and a
variable region sequence present in a cloned cDNA are ligated to a
sequence coding for a human antibody constant region followed by
ligation to a mammalian cell expression vector. The mouse leader
sequence may comprise, for example, the signal sequences shown in
FIG. 1 or FIG. 2.
[0069] The polypeptide comprising human antibody constant region
can comprise any of the heavy or light chain constant regions of
human antibodies, including, for example, .gamma.1, .gamma.2,
.gamma.3 or .gamma.4 for heavy chains, and .kappa. for light
chains.
[0070] To prepare a chimeric antibody, two expression vectors are
constructed. A first expression vector contains DNAs coding for the
light chain variable region and human light chain constant region
under the control of an expression control element such as an
enhancer/promoter system. A second expression vector contains DNAs
coding for the heavy chain variable region and human heavy chain
constant region under the control of an expression control element
such as an enhancer/promoter system. Host cells such as mammalian
cells (for example, COS cell) are cotransformed with these
expression vectors. The transformed cells are cultivated in vitro
or in vivo to produce a chimeric antibody, See, e.g.
WO91/16928.
[0071] As an alternative, mouse leader sequence present in cloned
cDNA and DNAs coding for mouse light chain variable region and
human light chain constant regions, as well as a mouse leader
sequence and DNAs coding for mouse heavy chain variable region and
human heavy chain constant region, are introduced into a single
expression vector (see, for example, WO94/11523). The single vector
is used to transform a host cell. The transformed host is cultured
in vivo or in vitro to produce a desired chimeric antibody.
[0072] The vector for the expression of the heavy chain of the
chimeric antibody can be obtained by introducing cDNA comprising a
nucleotide sequence coding for the mouse heavy chain variable
region into a suitable expression vector containing genomic DNA
comprising a nucleotide sequence coding for the heavy chain
constant region of human antibody, or cDNA coding for the heavy
chain constant region. As indicated above, the heavy chain constant
region may comprise, for example, .gamma.1, .gamma.2, .gamma.3 or
.gamma.4.
[0073] Expression vectors comprising genomic DNA coding for a heavy
chain constant region include, for example, HEF-PMh-g gamma 1
(WO92/19759) and DHER-INCREMENT E-RVh-PM1-f (WO92/19759).
Alternatively, a human constant region library can be prepared
using cDNA from human peripheral blood mononuclear cells, as
described by Liu. et al., Proc. Natl. Acad. Sci. USA, 84:3439-43
(1987) or Reff et al., Blood 83(2): 435-45 (1994), for example.
[0074] The cDNA coding for the mouse heavy chain variable region of
SEQ ID NO:11 (e.g., the nucleotide sequence SEQ ID NO:23, which
does not contain a signal sequence segment; or the nucleotide
sequence of SEQ ID NO:25, which contains a signal sequence) is
treated with suitable restriction enzyme(s) and inserted into
genomic DNA coding for a heavy chain constant region, to construct
a chimeric heavy chain expression vector containing the genome DNA
coding for the heavy chain constant region. Insertion is by
ligation of the cDNA encoding the heavy chain constant region, and
insertion into an expression vector such as pQCXIH (Clontech), to
construct an expression vector containing the complete cDNA
encoding the complete chimeric heavy chain. Alternatively, the cDNA
encoding the mouse heavy chain variable region may be ligated into
an appropriate commercially available cloning plasmid that
expresses the constant region of a human heavy chain, e.g.,
pFUSE-CHIg-hG1 (InvivoGen, San Diego, Calif.).
[0075] In a similar fashion, a vector for the expression of the
light chain of the chimeric antibody can be constructed by ligating
a cDNA coding for the mouse light chain variable region (e.g., the
nucleotide sequence SEQ ID NO:22, which does not contain a signal
sequence; or the nucleotide sequence of SEQ ID NO:24, which
contains a signal sequence) and a genomic DNA or cDNA coding for
the light chain constant region of a human antibody, and
introduction into a suitable expression vector. The light chain
constant region includes, for example, .kappa. or .lamda. chains.
Any of the four known .lamda. chain constant region isotypes may be
utilized. Alternatively, the cDNA encoding the mouse light chain
variable region may be ligated into an appropriate commercially
available cloning plasmid that expresses the constant region of a
human .kappa. light chain, e.g., pFUSE2-CLIg-hk (InvivoGen, San
Diego, Calif.).
[0076] FIG. 11 shows the DNA (SEQ ID NO:20) and amino acid (SEQ ID
NO:8) sequences of a construct encoding a mouse heavy chain
variable region (mV.sub.H) containing a leader signal peptide, for
the preparation of a further construct for expressing a chimeric
antibody heavy chain consisting of the mV.sub.H and the constant
region of a human .gamma. chain (mV.sub.H-h.gamma.). FIG. 12 is a
schematic of a plasmid for expression of the mV.sub.H-h.gamma.,
utilizing the mV.sub.H construct of FIG. 11. The mV.sub.H construct
was cloned into the pFUSE-CHIg-hG1 plasmid as shown in FIG. 12.
Restriction sites utilized in the cloning strategy are
indicated.
[0077] FIG. 13 shows the DNA (SEQ ID NO:21) and amino acid (SEQ ID
NO:7) sequences of a construct encoding a mouse light chain
variable region (mV.sub.L) containing a leader signal peptide, for
the preparation of a plasmid for expressing a chimeric antibody
heavy chain consisting of the mV.sub.L and the constant region of a
human .kappa. chain (mV.sub.L-h.kappa.). FIG. 14 is a schematic of
a plasmid for expression of the chimeric mV.sub.L-h.kappa.,
utilizing the mV.sub.L construct of FIG. 13. The mV.sub.L construct
was cloned into the pFUSE2-CLIg-hk plasmid as shown in FIG. 14.
Restriction sites utilized in the cloning strategy are
indicated.
[0078] In another embodiment, the engineered antibody is a
humanized antibody. The humanized antibody comprises a human
antibody framework region and advantageously further comprises a
human antibody constant region. The six complementarity determining
regions (CDRs) SEQ ID NOS:1-6 are grafted to a human antibody. The
general genetic recombination procedure for producing humanized
antibodies are described, for example, in EP 125023 and WO
96/02576. Accordingly, a DNA sequence is designed in which DNA
encoding the aforementioned CDRs are ligated through framework
regions. The DNA sequence is synthesized by a polymerase chain
reaction method using oligonucleotide primers which are designed to
have regions overlapping the terminal regions of the CDRs and the
framework regions. The resultant DNA is ligated to DNA encoding the
human antibody constant region, and the ligation product is
integrated into an expression vector. The resultant recombinant
expression vector is introduced into a host, thereby producing the
humanized antibody. See, e.g., WO 96/02576.
[0079] The framework regions ligated through the CDRs are selected
so that the CDRs can form a functional antigen binding site. If
necessary, an amino acid(s) in the framework regions of the
antibody variable region may be replaced so that the CDRs of the
resulting humanized antibody can form an appropriate antigen
binding site. See Sato et al., Cancer Res. 53:851-6 (1993).
[0080] The amino acid sequences of the framework regions are
preferably selected to reflect a high homology to the framework
sequences of a human antibody. In this regard, a comparison may be
undertaken between the variable regions SEQ ID NO:10 and SEQ ID
NO:11 and the variable regions of structurally elucidated human
antibodies using, e.g., the Protein Data Bank. Other research tools
that may consulted include the Kabat Database of Sequences of
Proteins of Immunological Interest,
www<<dot>>kabatdatabase<<dot>>com and the
search tools included as part of that database; and Kabat, E. A. et
al., (1991) Sequences of Proteins of Immunological Interest,
5.sup.th edition. U.S. Department of Health and Human Services.
[0081] A humanized antibody variable region is selected as a basis
for the humanized antibody variable region. For example, the
framework region of a human antibody variable region having a high
homology, e.g., greater than about 80%, or greater than about 90%,
or greater than about 95%, with the framework regions of SEQ ID
NOS:10 and 11 (the non-CDR regions of SEQ ID NOS:10 and 11) is
selected. A polypeptide comprising the framework regions of the
humanized antibody and the CDRs of SEQ ID NOs:1-6 can be produced
by "CDR-grafting", a PCR-based method utilizing a DNA fragment of a
human antibody as a template. For an example of CDR grafting, see
Kettleborough et al., Protein Eng. 4(7):773-783 (1991); "Antibody
Humanization by CDR Grafting", Methods of Molecular Biology,
248(11):135-159 (2004).
[0082] Humanized antibodies may be prepared, as exemplified in
Jones et al., 1986 Nature 321:522-525, which describes replacing
the complementarity-determining regions in a human antibody with
those from a mouse. Also see Riechmann, 1988, Nature 332:323-327;
Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86:10029 (preparation
of humanized antibody binding the interleukin 2 receptor); and
Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA 86:3833
(describing the cloning of immunoglobulin variable domains for
expression by the polymerase chain reaction).
[0083] Any suitable expression system may be used to produce the
chimeric or humanized antibody. For example, eukaryotic cells
include animal cells such as established mammalian cell lines,
fungal cells, and yeast cells; prokaryotic cells include bacterial
cells such as Escherichia coli. Mammalian host cells are preferred.
The expression system may incorporate conventional promoters useful
for the expression in mammalian cells, e.g., the human
cytomegalovirus (HCMV) immediate early promoter. Promoters for
expression in mammalian cells may include virus promoters, such as
those of retrovirus, polyoma virus, adenovirus and simian virus
(SV) 40, and mammalian cell derived promoters, such as those of
human polypeptide chain elongation factor-1 alpha (HEF-1
alpha).
[0084] The expression system may include a replication origin such
as those derived from SV40, polyoma virus, adenovirus or bovine
papilloma virus (BPV). The expression vector may comprise a gene
for phosphotransferase APH(3') II or I (neo), thymidine kinase
(TK), E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt)
or dihydrofolate reductase (DHFR) as a selective marker for
increasing the gene copy number in a host cell system.
[0085] The expressed chimeric or humanized antibody is produced by
culturing the thus-transformed host cells, and isolating and
purifying the antibody from the cells according to well-known
techniques. The concentration of the resulting purified antibody
can be determined by, for example, enzyme-linked immunosorbent
assay (ELISA). Antigen-binding activity can be confirmed by known
methods antibody, techniques such as ELISA, enzyme immunoassay,
radioimmunoassay or fluorescent assay.
[0086] Fragments of the chimeric or humanized antibody retaining
antigen-binding activity may be prepared, e.g., Fab, F(ab').sub.2,
and Fv fragments. Antibody fragments can be produced by cleaving
the antibody with an enzyme (e.g., papain, pepsin) into antibody
fragments, or by constructing a gene encoding the antibody fragment
and inserting the gene into an expression vector and introducing
the resultant recombinant expression vector into a suitable host
cell, thereby expressing the antibody fragment (see, for example,
Co et al., J. Immunol. 152:2968-76 (1994)).
[0087] In another embodiment, the engineered antibody is a
single-chain antibody (SCA), also referred to herein as a
single-chain variable fragment (scFv), comprising a light chain
variable region comprising the complementarity determining regions
(CDRs) having the amino acid sequences shown in SEQ ID NO:1, SEQ ID
NO:2 and SEQ ID NO:3, and a heavy chain variable region comprising
the complementarity determining regions having the amino acid
sequences shown in SEQ ID NO:5 and SEQ ID NO:6. The scFv can be
produced by ligating a heavy chain variable region comprising the
CDRs of SEQ ID NOS: 5 and 6 to a light chain variable region
comprising the CDRs of SEQ ID NOS:1, 2 and 3 through a linker. The
linker is preferably a peptide linker, i.e., the linker is composed
of amino acid residues. The residues for the linker may be selected
from naturally occurring amino acids, non-naturally occurring amino
acids, and modified amino acids. The linker will typically connect
the carboxy terminus of the heavy chain variable region to the
amino terminus of said light chain variable region. The reverse is
also possible, i.e., using the linker to connect the carboxy
terminus of the light chain variable region to the amino terminus
of the heavy chain variable region. The linker may comprise any
number of amino acids. The linker may thus comprise, for example,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, or more amino acids. In some embodiments,
the linker may be composed of from 3 to 60 amino acid residues,
from 3 to 40 amino acids, from 3 to 30 amino acids, from 3 to 24
amino acids, from 3 to 18 amino acids, or from 3 to 15 amino acids.
The linker may comprise, for example, a repeating sub-sequence of
2, 3, 4, 5 or more amino acid residues, comprising 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12 or more repeats of the sub-sequence.
[0088] In one embodiment, the linker comprises the amino acid
sequence Gly-Ser, or repeats thereof. See, e.g., Huston, et al.,
Methods in Enzymology, 203:46-88 (1991). In another embodiment, the
linker comprises the amino acid sequence Glu-Lys, or repeats
thereof. See, e.g., Whitlow et al., Protein Eng., 6:989 (1993)). In
another embodiment, the linker comprises the amino acid sequence
Gly-Gly-Ser, or repeats thereof. In another embodiment, the linker
comprises the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:29), or repeats thereof. In certain specific embodiments, the
linker contains from 2 to 12 repeats of Gly-Gly-Ser or
Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29).
[0089] An scFv of the invention having the amino acid sequence SEQ
ID NO:14 is illustrated in FIG. 3. The scFv comprises from amino
terminus to carboxy terminus, (i) an initial methionine residue,
(ii) the mV.sub.L sequence SEQ ID NO:9, which comprises the
parental mV.sub.L of SEQ ID NO:10 without the terminal Arg residue
of SEQ ID NO:10, (iii) Val-Asp, (iv) a linker, (v) Leu-Glu, (vi)
the mV.sub.H sequence SEQ ID NO.11, and (vii) a FLAG-Tag for
affinity chromatography purification.
[0090] The antibody or antibody fragments of the invention may be
utilized to bind to the C-terminal telopeptides of the
.alpha.2-chain of collagen to inhibit the formation of collagen
fibrils, and thereby prevent excessive deposition of collagen
fibrils that is characteristic of fibrotic processes. The antibody
or antibody fragments of the invention may thus be utilized to
reduce localized and systemic fibrotic lesions, including but not
limited to fibroses occurring in internal organs, the dermus or the
eye.
[0091] Types of fibrosis that may be treated or prevented include,
for example, pulmonary fibrosis, idiopathic pulmonary fibrosis,
cirrhosis, endomyocardial fibrosis, mediastinal fibrosis,
myelofibrosis, retroperitoneal fibrosis, progressive massive
fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid
formation, myocardial infarction, scleroderma/systemic sclerosis,
arthrofibrosis, and adhesive capsulitis. The fibrotic lesion
treated may be triggered by trauma, accidental injury or surgical
procedures, among other causes. The antibody or antibody fragments
of the invention may be administered, for example, after surgery in
the abdomen to avoid the formation of excessive scar tissue around
abdominal organs; after plastic surgery to the face to reduce scar
formation; to the eye following glaucoma surgery performed to
maintain a lamellar channel from the subconjunctival space to the
anterior chamber, to prevent excessive scar formation that may
function to closes the pressure-reducing channel and cause
intraocular pressure to rise; and following implantation of medical
devices and materials implanted in the human body, which would
otherwise trigger a fibrotic response. In one embodiment, the
antibody or antibody fragments are administered to teat or prevent
the formation of keloids,
[0092] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more pharmaceutically acceptable carriers or excipients. The
antibodies or fragments thereof can be formulated for
administration in accordance with the route of administration.
Formulations for injection can be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions can take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and can
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the antibody can be in
lyophilized powder form for constitution with a suitable vehicle,
e.g., sterile pyrogen-free water, before use.
[0093] In some embodiments, the subject of treatment is human. In
other embodiments, the subject is a veterinary subject.
[0094] Treatment may involve administration of one or more
antibodies or antibody fragments of the invention, alone or with a
pharmaceutically acceptable carrier. The active agent may be
administered in combination with one or more other therapeutic,
diagnostic or prophylactic agents. The administered composition may
thus further comprise an additional agent selected from the group
consisting of corticosteroids, antiinflammatories,
immunosuppressants, antimetabolites, and immunomodulators, for
example.
[0095] The pharmaceutically acceptable carrier may comprise any
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption enhancing or delaying agents, and
the like that are physiologically compatible. Some examples of
pharmaceutically acceptable carriers are water, saline, phosphate
buffered saline, acetate buffer with sodium chloride, dextrose,
glycerol, polyethylene glycol, ethanol and the like, as well as
combinations thereof. In some cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Additional examples of pharmaceutically acceptable substances are
surfactants, wetting agents or minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
antibody.
[0096] The compositions used in the practice of the invention may
be in a variety of forms, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions, dispersions or suspensions,
tablets, pills, lyophilized cake, dry powders, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application. In some embodiments,
the antibody or antibody fragment may be administered by using a
pump, enema, suppository, or indwelling reservoir or such like.
[0097] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, lyophilized cake, dry
powder, microemulsion, dispersion, liposome, or other ordered
structure suitable to high drug concentration. Sterile solutions
can be prepared by incorporating the antibody or fragment in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by
sterilization. In the case of sterile powders for the preparation
of sterile solutions, the preferred methods of preparation are
vacuum drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile solution thereof. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. The desired
characteristics of a solution can be maintained, for example, by
the use of surfactants and the required particle size in the case
of dispersion by the use of surfactants, phospholipids and
polymers. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts, polymeric materials,
oils and gelatin.
[0098] In certain embodiments, the antibody or antibody fragment
compositions of the invention may be prepared with a carrier that
will protect the antibody against rapid release, such as a
controlled release formulation, including implants, transdermal
patches, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York
(1978)).
[0099] The compositions of the invention may be administered
parenterally. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion
techniques.
[0100] The compositions of the invention may be given orally, in
any orally acceptable dosage form including, but not limited to,
capsules, tablets, aqueous suspensions or solutions. In the case of
tablets for oral use, carriers which are commonly used include
lactose and corn starch. Lubricating agents, such as magnesium
stearate, are also typically added. For oral administration in a
capsule form, useful diluents include lactose and dried corn
starch.
[0101] The compositions of the invention may be administered
topically. For topical applications, the pharmaceutical
compositions may be formulated in a suitable ointment containing
the active component suspended or dissolved in one or more
carriers. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Topically-transdermal patches may also be used. For topical
applications, the pharmaceutical compositions may be formulated in
a suitable ointment containing the active component suspended or
dissolved in one or more carriers. Carriers for topical
administration of the compounds of this invention include, but are
not limited to, mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene, polyoxypropylene compound,
emulsifying wax and water. Alternatively, the pharmaceutical
compositions can be formulated in a suitable lotion or cream
containing the active components suspended or dissolved in one or
more pharmaceutically acceptable carriers. Suitable carriers
include, but are not limited to, mineral oil, sorbitan
monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol and water.
[0102] Topical or other localized administration is advantageously
utilized at the site of a localized fibrosis, e.g., by localized
injection. The location of the fibrosis may comprise, for example,
a wound, particularly a wound edge.
[0103] The dosage of active agent may vary according to factors
such as the disease state, age, sex, and weight of the individual,
and the ability of the antibody or antibody portion to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
antibody or antibody portion are outweighed by the therapeutically
beneficial effects. Detection and measurement of indicators of
efficacy may be measured by a number of available diagnostic tools,
including, for example, by physical examination including blood
tests, pulmonary function tests, and chest X-rays; CT scan;
bronchoscopy; bronchoalveolar lavage; lung biopsy and CT scan. A
"prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired prophylactic result. Typically, since a prophylactic dose
is used in subjects prior to or at an earlier stage of disease, the
prophylactically effective amount may be less than the
therapeutically effective amount.
[0104] Dosage regimens can be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus can be administered, several divided
doses can be administered over time or the dose can be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a pre-determined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0105] As a non-limiting example, an effective amount of an
antibody or antibody fragment active agent is from about 0.025 to
about 50 mg/kg, or from about 0.1 to about 50 mg/kg, or from about
0.1-25 mg/kg, or from about 0.1 to about 10 mg/kg, or from about
0.1 to about 3 mg/kg. Dosage may vary with the type and severity of
the condition to be alleviated. For any particular treatment
subject, specific dosage regimens may be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0106] The practice of the invention is illustrated by the
following non-limiting example. The invention should not be
construed to be limited solely to the compositions and methods
described herein, but should be construed to include other
compositions and methods as well. One of skill in the art will know
that other compositions and methods are available to perform the
procedures described herein.
EXAMPLES
Example 1: Single-Chain Antibody by Bacterial Expression
[0107] A construct for preparing a single chain antibody, also
referred to herein as a single-chain variable fragment (scFv), was
prepared containing DNA sequences encoding the light chain variable
region of SEQ ID NO:7 and the heavy chain variable region of SEQ ID
NO:8. The DNA sequences were obtained by PCR from a hybridoma
expressing an IgA class antibody to collagen .alpha.2-chain (Chung
et al., J. Biol. Chem. 283(38):25879-25886 (2008)). Total RNA was
prepared from hybridoma cells with the use of an RNA-isolation kit
according to the manufacturer's protocol (QIAGEN). PCR products
spanning the V.sub.H of the .alpha. and the V.sub.L of the .kappa.
chains were cloned into the pETBlue-1 Blunt vector and
sequenced.
[0108] A. Bacterial Expression System
[0109] For the bacterial expression of the scFv, a construct was
created in which the V.sub.L region was connected with the V.sub.H
region via a 15 amino acid linker comprising three repeats of the
Gly-Gly-Gly-Gly-Ser (SEQ ID NO:29) motif. A DNA sequence encoding a
FLAG tag was fused to the 3' end of the construct to facilitate
downstream purification of the recombinant scFv (FIG. 3). Minor
additions to the original amino acid sequences of the V.sub.L (SEQ
ID NO:10) and V.sub.H (SEQ ID NO:11) regions were created as a
consequence of the cloning process (FIG. 3). Moreover, the native
signal peptides shown in FIGS. 1 and 2 (single underlining) were
omitted in the scFv construct. The constructs were thus arranged to
provide a scFv comprising, from N-terminus to C-terminus, light
chain variable region--linker--heavy chain variable region, where
the linker connected the N-terminus of the heavy chain to the
C-terminus of the light chain. More specifically, the scFv
comprised, from amino terminus to carboxy terminus, (i) an initial
methionine residue, (ii) the mV.sub.L sequence SEQ ID NO:9, which
comprises the parental mV.sub.L of SEQ ID NO:10 without the
terminal Arg residue of SEQ ID NO:10, (iii) Val-Asp, (iv) a linker,
(v) Leu-Glu, (vi) the mV.sub.H sequence SEQ ID NO.11, and (vii) a
FLAG-Tag. The fidelity of the construct was confirmed by DNA
sequencing.
[0110] The scFv DNA construct was initially cloned into the
NcoI/SacI site of the pET-28 (not shown), a vector that does not
include any signaling sequences for the secretion of exogenous
proteins into the periplasm (EMD Biosciences). Because the
expression of exogenous proteins in bacteria is frequently
associated with the formation of insoluble aggregates packed into
the inclusion bodies, the pET-22 vector was also employed (FIG. 4)
(EMD Biosciences). In contrast to the pET-28 vector, the pET-22
vector carries an N-terminal pelB signal sequence for potential
periplasmic localization of the expressed protein, a situation in
which the produced proteins are potentially more soluble. The two
above vectors were tested for their ability to facilitate the
production of the scFv variant.
[0111] Bacteria expressing the scFv cloned into the pET-22 and
pET-28 vectors were cultured in the presence of ampicillin or
kanamycin, respectively. After reaching the optical density
(OD.sub.600) of 0.5 units, bacterial cultures were treated with 1
mM isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) to induce the
expression of the scFv. Subsequently, the bacterial cultures were
incubated for an additional 3 h at 37.degree. C.
[0112] Bacteria were collected by centrifugation and then lysed
with the B-PER II Protein Extraction Reagent (Thermo Scientific) in
the presence of lysozyme, and then the scFv-rich insoluble
inclusion bodies were collected by centrifugation. Subsequently,
the inclusion bodies were solubilized in 6M guanidinium
hydrochloride (GdnHCl) in the presence of the reducing agent
dithiothreitol (DTT). Refolding conditions were tested by running
pilot-scale refolding assays with the use of the Protein Refolding
Kit (Thermo Scientific). As a result of these assays, the denatured
scFv molecules present in the GdnHCl-solubilized material were
refolded by diluting them into 100 volumes of a refolding buffer
consisting of 55 mM Tris, 21 mM NaCl, 0.88 mM KCL, pH 8.2.
Subsequently, the sample was concentrated 100-fold by
ultrafiltration.
[0113] B. scFv Purification and Analyses
[0114] Pilot purification of the scFv was carried out with the
agarose-conjugated anti-FLAG antibody according to the
manufacturer's protocol (Sigma-Aldrich). The purity of purified
soluble scFv was analyzed by polyacrylamide gel electrophoresis and
by Western blot assay with the anti-FLAG antibody (Sigma-Aldrich)
run in denaturing and reducing conditions. The result is shown in
FIG. 5A. The electrophoretic migration of the purified scFv
indicated the predicted mass.
[0115] In addition, due to the relatively high cost of the
anti-FLAG antibody, an ion exchange chromatography on the MonoQ
Sepharose resin (GE Healthcare Life Sciences) was employed to
purify the semi-preparative amounts of the scFv variant. In brief,
a sample containing the refolded scFv was loaded onto a MonoQ
Sepharose column, and then bound proteins were eluted with a 0-1 M
NaCl gradient. The collected peaks were then checked for the
presence of the scFv variant by gel electrophoresis assays and
Western blot assays with the use of the anti-FLAG antibody
(Sigma-Aldrich) (FIG. 5B) in denaturing and reducing conditions.
The fraction including the scFv was eluted with 200 mM NaCl, while
contaminating proteins were eluted with 500 mM NaCl (FIG. 5B).
[0116] The molecular mass of the purified native scFv was analyzed
by high pressure liquid size exclusion chromatography (SEC HPLC)
run in non-denaturing conditions. The chromatography profile is
shown in FIG. 6. The three main peaks in FIG. 6 indicate the single
chain antibody monomers and oligomers.
[0117] The binding specificity of the scFv for its intended
epitope, the .alpha.2-chain of collagen, was confirmed by a Western
blot-based assay as follows. Purified human procollagen I was
electrophoresed in a polyacrylamide gel. Subsequently, procollagen
chains were transferred to a nitrocellulose membrane. Next, the
membranes were incubated with FLAG-tagged single chain antibody.
The bound single chain antibody was detected with chemiluminescence
by an anti-FLAG antibody conjugated to horseradish peroxidase. The
results are shown in FIG. 7. The lines in FIG. 7 depict different
repeats of the assay, and were identified as procollagen I .alpha.2
chains, thereby indicating specific binding of the scFv to collagen
.alpha.2-chain.
Example 2: Single Chain Antibody Inhibition of Collagen Fibril
Deposition
[0118] Assays of the inhibition of collagen fibril deposition by
the scFv of Example 1 were done in cultures of keloid-derived
fibroblasts. In brief, the fibroblasts were seeded into the wells
of a 96-well plate at the density of 4.times.10.sup.3 cells/well.
Six hours after seeding, the attached cell layers were washed and
fresh media supplemented with 40 .mu.g/ml of L-ascorbic acid
phosphate magnesium salt n-hydrate (Wako Pure Chemical Co.) was
added to the cells. Three experimental groups of cells were
analysed: (I) non treated, (II) treated with tissue growth factor
.beta. (TGF-.beta.) and the scFv, and (III) treated only with
TGF-.beta.. TGF-.beta. was added at a concentration of 1 ng/ml,
while scFV was added at 1, 5 or 10 .mu.g/ml. The purpose of adding
TGF-.beta., an activator of collagen biosynthesis, was to stimulate
cells to produce and secrete high amounts of collagen. After the
48-h culture in the presence or the absence of TGF-.beta. and scFv,
layers consisting of collagen-rich extracellular matrix and cells
were solubilized by adding a lysis buffer and analyzed by Western
blot assays for the presence of collagen I, a main constituent of
collagen fibrils. The results shown in FIG. 8 indicate a marked
decrease of collagen deposits in a group treated with the scFv. An
equal number of cells in the analyzed groups is indicated by
comparable amounts of GAPDH. The analyzed groups and the collagen I
marker (Cm) are indicated.
Example 3: Constructs for Single-Chain Antibody Production by Yeast
Expression
[0119] Constructs for the expression of the scFv of Example 1 in
yeasts were also engineered. The rationale for employing yeasts to
produce the scFv variants was dictated by the fact that those
organisms are eukaryotes, a characteristic that potentially may
improve the folding and solubility of the recombinant scFv
variants.
[0120] Accordingly, the following scFv variants were designed: (i)
A_L_K, a construct in which a cassette for the V.sub.H of the mouse
heavy .alpha. chain was linked with a cassette for the V.sub.L of
the mouse light .kappa. chain; and (ii) K_L_A, a construct in which
a cassette for the V.sub.L of the light .kappa. chain was linked
with a cassette for the V.sub.H of the heavy .alpha. chain. In
comparison to the 15-amino acid linker designed for the bacterial
scFv construct (FIG. 3), the scFv constructs for yeast expression
consisted of a 30-amino acid linker. See FIGS. 9A-9C. By increasing
the length of the linker, the predicted solubility of the A_L_K and
K_L_A scFv variants will be higher than that with a shorter linker.
Moreover, the increased length of the linker may decrease the
potential of scFv variants to form oligomers. If necessary, that
the length of the linker may be readily changed by DNA engineering
technology.
[0121] The nucleotide (SEQ ID NOS:19 and 30) and amino acid (SEQ ID
NO:18) sequences of the A_L_K construct are shown in FIGS. 9A-9C. A
schematic of the same construct cloned into the pPIC-9K plasmid is
shown in FIG. 10. In both figures, restrictions sites utilized in
the cloning strategy are indicated--SnaBI and NotI restriction
sites were incorporated at the 5' and 3' ends of the construct,
respectively, to facilitate its downstream cloning into the pPIC-9K
yeast-expression vector (Invitrogen). A construct for the K_L_A
scFv was prepared in a similar way (not shown). The entire DNA
constructs for the A_L_K and K_L_A variants were synthesized
commercially (Blue Heron Biotechnology, Bothell, Wash. 98021 USA).
In both constructs, a His-tag-coding sequence was incorporated to
facilitate the downstream purification process (FIGS. 9A-9C). The
fidelity of constructs was confirmed by sequencing. The nucleotide
and amino acid sequences of the V.sub.H-linker-V.sub.L portion of
the A_L_K construct (without added restriction sites and His-Tag),
comprise SEQ ID NO:17 (nucleotide) and SEQ ID NO:16 (amino
acid).
Example 4: Preparation of Chimeric Antibody by Mammalian Cell
Expression
[0122] Constructs for the mammalian-cell expression of full-length
mouse/human chimeric IgG were created. The constructs consist of
sequences encoding a mouse variable region fused to sequences
encoding constant regions of human to .gamma. and .kappa. chains.
Two such DNA constructs were prepared as follows for the expression
of a chimeric mouse/human antibody. A first construct encoded a
chimera of the mouse-derived heavy chain variable region and a
human-derived heavy .gamma. chain (mV.sub.H-h.gamma.), while the
second construct encoded a chimera of the mouse-derived light chain
variable region and a human-derived light .kappa. chain
(mV.sub.L-h.kappa.).
[0123] A. Preparation of DNA Construct for Chimeric Mouse
V.sub.H-Human Heavy .gamma. Chain (mV.sub.H-h.gamma.)
[0124] The DNA sequence encoding the V.sub.H region of the heavy
mouse .alpha. chain of the original anti-.alpha.2Ct IgA was cloned
into the pETBlue-1 Blunt vector (EMD Biosciences/Novagen). To
facilitate downstream cloning into the pFUSE-CHIg-hG1 vector that
includes the sequence encoding the constant region (CH) of the
heavy .gamma. chain of the human IgG1 (InvivoGen), the EcoRI and
NheI restriction sites were introduced via PCR to the DNA sequence
encoding the V.sub.H region. See FIG. 11. (The nucleotide and amino
acid sequences shown in FIG. 11 are SEQ ID NO:20 and SEQ ID NO:8,
respectively.) Subsequently, an insert encoding the V.sub.H region
of the heavy mouse .alpha. chain was cloned into corresponding
restriction sites of the pFUSE-CHIg-hG1 vector (FIG. 12). The
fidelity of the mV.sub.H-h.gamma. construct was confirmed by DNA
sequencing.
[0125] B. Preparation of DNA Construct for Chimeric Mouse
V.sub.L-Human Light .kappa. Chain (mV.sub.L-h.kappa.)
[0126] DNA sequence encoding the V.sub.L region of the light mouse
.kappa. chain of the original anti-.alpha.2Ct IgA was cloned into
the pETBlue-1 Blunt vector (EMD Biosciences/Novagen). To facilitate
downstream cloning into the pFUSE2-CLIg-hk vector that includes the
sequence encoding the constant region (CH) of the human light
.kappa. chain (InvivoGen), the AgeI and BsiWI restriction sites
were introduced via PCR to the DNA sequence encoding the V.sub.L
region of the light mouse .kappa. chain. See FIG. 13. (The
nucleotide and amino acid sequences shown in FIG. 13 are SEQ ID
NO:21 and SEQ ID NO:7, respectively.) Subsequently, the insert
encoding the V.sub.L region of the light mouse .kappa. chain was
cloned into the corresponding restriction sites of the
pFUSE2-CLIg-hk vector (FIG. 14). The fidelity of the
mV.sub.L-h.kappa. construct was confirmed by DNA sequencing.
[0127] C. Selection of Clones Expressing Chimeric mV.sub.H-h.gamma.
and mV.sub.L-h.kappa. Variants in Mammalian Cells
[0128] Chimeric variants were expressed in Chinese hamster ovary
cells (CHO). In brief, CHO cells were transfected with a DNA
construct encoding the mV.sub.H-h.gamma. variant. Zeocin-resistant
clones were selected and screened for the presence of the
mV.sub.H-h.gamma.. Specifically, proteins secreted to the media by
selected clones were analyzed by Western blot for the presence of
the mV.sub.H-h.gamma. variant with the use of the goat anti-human
.gamma. chain polyclonal antibody conjugated with HRP
(Sigma-Aldrich). Next, the selected .gamma. chain-positive CHO
clone was expanded in cell culture and then transfected with a DNA
construct encoding the mV.sub.L-h.kappa. variant. Subsequently,
cell culture media from the double-transfected clones resistant to
Zeocin and Blasticidin were analyzed by Western blot for the
production of the mV.sub.L-h.kappa. chain with the use of the
polyclonal goat anti-human .kappa. chain antibodies conjugated with
HRP. Selected clones stably co-expressing mV.sub.H-h.gamma. and
mV.sub.L-h.kappa. chains were expanded in cell culture and then
cryopreserved in liquid nitrogen.
[0129] D. Purification of the Chimeric Antibody Consisting of the
mV.sub.H-h.gamma. and mV.sub.L-h.kappa. Chains
[0130] Cell culture media from selected CHO cells producing the
chimeric antibody were collected. Subsequently, proteins secreted
to the media were precipitated with ammonium sulfate added to a
50-% saturation. Precipitated proteins were collected by
centrifugation and then the protein pellet was solubilized in
phosphate buffered saline (PBS). Insoluble material was removed by
centrifugation while the supernatant was collected for further
processing. The chimeric IgG was purified by employing the
Protein-L agarose (Thermo Scientific), a resin that specifically
binds to the .kappa. chain of human but not bovine origin.
[0131] Purified chimeric antibodies were analyzed in reducing
conditions for the presence of the .gamma. and .kappa. chains.
Specific antibodies against particular chains confirm the
production of the .gamma. and .kappa. chains by CHO cells (FIG.
15A). To determine if the two chains of the chimeric IgG antibody,
i.e., mV.sub.H-h.gamma. and mV.sub.L-h.kappa. chains, co-assemble
into native-like molecules consisting of both types of chains
linked via disulfide bonds, the purified proteins secreted from CHO
cells were separated in a polyacrylamide gel in non-reducing
conditions. Subsequent Western blot assays with the anti-human
.gamma. chain and the anti human .kappa. chain antibodies indicate
that mV.sub.H-h.gamma. and mV.sub.L-h.kappa. chains were covalently
linked via disulfide bonds, thereby indicating the formation of
chimeric antibody molecules consisting of heavy and light chains of
expected molecular mass (FIG. 15B.). The presence of a high
molecular band indicates production of chimeric IgG molecules by
CHO cells. Human IgG with the .kappa. chain was used as a positive
marker (h-IgG(.kappa.).
[0132] E. Binding of the Chimeric IgG to its Designated Target
[0133] Western blot assays were employed to test the binding of
purified chimeric IgG to its designated target, i.e., the C
terminal telopeptide of the .alpha.2 chain of human procollagen I
(.alpha.2Ct). In brief, the .alpha.1 and .alpha.2 chains of
procollagen I purified from the cultured of human dermal
fibroblasts were separated in a polyacrylamide gel followed by
their transfer into a nitrocellulose membrane. Subsequently, the
membrane was incubated with chimeric IgG solubilized in a blocking
buffer. The binding of the chimeric IgG to the .alpha.2Ct was
detected by chemiluminescence with the use of the anti-human
.gamma. antibodies conjugated with horseradish peroxidase (HRP), as
shown in FIG. 16. Positive bands were identified as those
corresponding to intact procollagen I .alpha.2 chain (upper band)
and its partially degraded form. Native human IgG with the .kappa.
chain was used as a positive marker (Bethyl Laboratories Inc.).
Example 5: Binding Kinetics of Anti-.alpha.2Ct Variants to Human
Procollagen
[0134] The binding of the anti-.alpha.2Ct variants (scFv and
chimeric-Ig) to human procollagen I was analyzed with the use of
the SensiQ Pioneer biosensor (ICx Nomadics). In brief, purified
human procollagen I, a protein that includes a native .alpha.2 C
telopeptide, was covalently immobilized on a sensor chip (COOH2,
ICx Nomadics). Subsequently, the kinetics of the binding of the
original anti-.alpha.2Ct mouse IgA antibody, the binding of the
bacterial-derived scFv variant of Example 1, and the binding of the
mouse-human chimeric IgG of Example 4, were analyzed. Human IgG
with the light .kappa. chain was used as a control (Bethyl
Laboratories, Inc.). The dissociation equilibrium constant
(K.sub.D) values for each interaction were calculated with the use
of the QDat software (ICx Nomadics). The results are show in FIG.
17. In each panel, the curves represent association and
dissociation events during the interaction between immobilized
procollagen I and free antibody variants present at concentrations
ranging from 4.times.10.sup.-7 M to 1.25.times.10.sup.-8 M. For
each assay, the association rate constants (k.sub.on) and the
dissociation rate constants (k.sub.off) were obtained, and the
equilibrium dissociation constants (K.sub.D) values were calculated
from a ratio of k.sub.off/k.sub.on. Although specific K.sub.D
values differ, all variants are characterized by a strong binding
affinity to procollagen I. The lack of binding of control human IgG
to procollagen I indicates the high specificity of the presented
binding assays.
Example 6: Expression, Purification and Testing of scFv Variants
Produced in Yeast
[0135] A. Expression of scFv in Yeasts
[0136] Yeast clones expressing the A_L_K or K_L_A scFv variants
cloned into the pPIC-9K yeast-expression vector according to
Example 3 were selected by culturing them in the absence of
histidine (His(-) conditions) according to manufacturer's
suggestions (Invitrogen). Subsequently, the selected clones were
tested for production of the scFv variants. In brief, the selected
clones were cultured in the presence of methanol as a sole source
of carbon. After six days cell culture media were tested for the
presence of secreted scFv variants. Specifically, His-tagged scFv
variants were detected by Western blot assays in which the anti-His
tag antibody was employed. In addition to Western blot assays, PCR
was employed to confirm the presence of DNA encoding the specific
scFv variants in the yeasts' genome.
[0137] The results are shown in FIGS. 18A (Western blot assay) and
18B (PCR assay). Because of noticeably smaller yield of the K_L_A
variant (not shown), only the A_L_K scFv construct was chosen for
further analyses. As shown in FIG. 18A, Western blot assays of the
A_L_K scFv variant secreted by various yeast clones cultured in His
(-) conditions stained positive for His-tagged scFv variant. Cell
culture media from non-transformed yeast cells (Ctrl) are negative
for the scFv variant. As shown in FIG. 148, PCR assay confirmed the
presence of DNA constructs encoding the A_L_K and K_L_A variants in
the genome of analyzed clones.
[0138] B. Purification of Yeast scFv Variants
[0139] Purification of the scFv variants was carried out with a
nickel column according to the manufacturer's protocol
(Invitrogen). The His-tagged A_L_K was purified on the nickel
column. The purified scFv variants were analyzed by gel
electrophoresis and by Western blot assays run in denaturing and
reducing conditions. Furthermore, the molecular mass of the
purified native scFv was analyzed by high pressure liquid size
exclusion chromatography (SEC HPLC) run in non-denaturing
conditions.
[0140] As indicated in FIG. 19, the A_L_K scFv variant eluted from
the nickel column contributed the main band seen in the
electrophoretic gel. In the denaturing/reducing conditions of the
assay, the A_L_K scFv variant purified from yeast cultures migrated
in the electrophoretic field according to its predicted mass of 28
kDa. SEC HPLC assays (not shown), however, demonstrate that in the
applied native conditions, most of the concentrated scFv molecules
(concentration of 0.6 mg/ml) aggregate to form high-molecular mass
assemblies. Such aggregate formation is a common characteristic of
recombinant proteins, and may be remedied by optimization of
experimental conditions in which aggregate formation is
minimized.
[0141] C. Western Blot Assays of scFv Binding to its Designated
Target
[0142] Western blot assays were employed to test the binding of
purified scFv to its designated target i.e. the C terminal
telopeptide of the .alpha.2 chain of human procollagen I
(.alpha.2Ct). In brief, the Pro-.alpha.1 and Pro-.alpha.2 chains of
procollagen I purified from the culture of human dermal fibroblasts
were separated in a polyacrylamide gel followed by their transfer
into a nitrocellulose membrane. Subsequently, the nitrocellulose
membrane was incubated with scFv. The binding of the His-tagged
scFv to the .alpha.2Ct was detected by chemiluminescence with the
use of the anti-His antibodies conjugated with horseradish
peroxidase (HRP). The results, shown in FIG. 20. Lane (A)
represents the electrophoresis of the purified human procollagen I
in a polyacrylamide gel (Coomassie blue-stained chains are
indicated). Lane B represents the nitrocellulose membrane incubated
with His-tagged yeast-derived scFv. The presented protein band
(Lane B) was identified as procollagen I .alpha.2 chain, thereby
indicating specific scFv-.alpha.2Ct binding.
[0143] The disclosures of each and every patent, patent
application, publication and GenBank record cited herein are hereby
incorporated herein by reference in their entirety.
[0144] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. While the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope used in
the practice of the invention. The appended claims are intended to
be construed to include all such embodiments and equivalent
variations.
Sequence CWU 1
1
30117PRTMus musculus 1Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr
Arg Lys Asn Asn Leu1 5 10 15Ala27PRTMus musculus 2Trp Ala Ser Thr
Arg Glu Ser1 538PRTMus musculus 3Lys Gln Ser Tyr Asn Leu Trp Thr1
5416PRTMus musculus 4Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu
Lys Lys Pro Gly Glu1 5 10 15519PRTMus musculus 5Met Ala Trp Ile Asn
Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp1 5 10 15Phe Thr
Gly65PRTMus musculus 6Gly Tyr Tyr Tyr Tyr1 57128PRTMus
musculusSIGNAL(1)..(15) 7Met Val Leu Met Leu Leu Leu Leu Trp Val
Ser Gly Thr Cys Gly Asp1 5 10 15Ile Val Met Ser Gln Ser Pro Ser Ser
Leu Ala Val Ser Ala Gly Glu 20 25 30Lys Val Thr Met Ser Cys Lys Ser
Ser Gln Ser Leu Leu Asn Ser Arg 35 40 45Thr Arg Lys Asn Asn Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ser 50 55 60Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val Pro65 70 75 80Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95Ser Ser Val
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln Ser 100 105 110Tyr
Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120
1258133PRTMus musculusSIGNAL(1)..(19) 8Met Gly Trp Val Trp Asn Leu
Leu Phe Leu Met Ala Ala Ala Gln Cys1 5 10 15Ile Gln Ala Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 20 25 30Pro Gly Glu Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Pro
Leu His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60Gln Trp Met
Ala Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala65 70 75 80Asp
Asp Phe Thr Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90
95Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr
100 105 110Tyr Phe Cys Val Arg Gly Tyr Tyr Tyr Tyr Trp Gly Gln Gly
Thr Thr 115 120 125Leu Ser Val Ser Ser 1309112PRTMus 9Asp Ile Val
Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly1 5 10 15Glu Lys
Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Arg
Thr Arg Lys Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
Cys Lys Gln 85 90 95Ser Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 11010113PRTMus musculus 10Asp Ile Val Met
Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly1 5 10 15Glu Lys Val
Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Arg Thr
Arg Lys Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55
60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65
70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys
Gln 85 90 95Ser Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110Arg11114PRTMus musculus 11Gln Ile Gln Leu Val
Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Pro Leu His
Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Gln Trp Met 35 40 45Ala Trp
Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60Thr
Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75
80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95Val Arg Gly Tyr Tyr Tyr Tyr Trp Gly Gln Gly Thr Thr Leu Ser
Val 100 105 110Ser Ser12246PRTArtificial SequenceRecombinantly
produced single chain antibody 12Met Asp Ile Val Met Ser Gln Ser
Pro Ser Ser Leu Ala Val Ser Ala1 5 10 15Gly Glu Lys Val Thr Met Ser
Cys Lys Ser Ser Gln Ser Leu Leu Asn 20 25 30Ser Arg Thr Arg Lys Asn
Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45Gln Ser Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 50 55 60Val Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu65 70 75 80Thr Ile
Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys 85 90 95Gln
Ser Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys Val Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Leu Glu Gln Ile Gln Leu Val Gln Ser Gly Pro Glu
Leu Lys 130 135 140Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Thr145 150 155 160Phe Thr Asp Tyr Pro Leu His Trp Val
Lys Gln Ala Pro Gly Lys Gly 165 170 175Leu Gln Trp Met Ala Trp Ile
Asn Thr Glu Thr Gly Glu Pro Thr Tyr 180 185 190Ala Asp Asp Phe Thr
Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala 195 200 205Ser Thr Ala
Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala 210 215 220Thr
Tyr Phe Cys Val Arg Gly Tyr Tyr Tyr Tyr Trp Gly Gln Gly Thr225 230
235 240Thr Leu Ser Val Ser Ser 24513738DNAArtificial
SequenceRecombinantly produced polynucleotide encoding single chain
antibody of SEQ ID NO12 13atggacattg tgatgtcaca gtctccatcc
tccctggctg tgtcagcagg agagaaggtc 60actatgagct gcaaatccag tcagagtctg
ctcaacagta gaacccgaaa gaataacttg 120gcttggtacc agcagaaacc
agggcagtct cctaaactgc tgatctactg ggcatccact 180agggaatctg
gggtccctga tcgcttcaca ggcagtggat ctgggacaga tttcactctc
240accatcagca gtgtgcaggc tgaagacctg gcagtttatt actgcaagca
atcttataat 300ctgtggacgt tcggtggagg caccaagctg gaaatcaaag
tcgacggtgg tggtggttct 360ggcggcggcg gctccggtgg tggtggttct
ctcgagcaga tccagttggt gcagtctgga 420cctgagctga agaagcctgg
agagacagtc aagatctcct gcaaggcttc tggttatacc 480ttcacagact
atccattgca ctgggtgaag caggctccag gaaagggttt acagtggatg
540gcctggataa acactgagac tggtgagcca acatatgcag atgacttcac
gggacggttt 600gccttctctt tggagacctc tgccagcact gcctatttgc
agatcaacaa cctcaaaaat 660gaggacacgg ctacatattt ctgtgttaga
ggttattatt actactgggg ccaaggcacc 720actctctcag tctcctca
73814254PRTArtificial SequenceRecombinantly produced single chain
antibody comprising C-terminal FLAG tag 14Met Asp Ile Val Met Ser
Gln Ser Pro Ser Ser Leu Ala Val Ser Ala1 5 10 15Gly Glu Lys Val Thr
Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 20 25 30Ser Arg Thr Arg
Lys Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45Gln Ser Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 50 55 60Val Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu65 70 75
80Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys
85 90 95Gln Ser Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 100 105 110Lys Val Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly 115 120 125Gly Ser Leu Glu Gln Ile Gln Leu Val Gln Ser
Gly Pro Glu Leu Lys 130 135 140Lys Pro Gly Glu Thr Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr145 150 155 160Phe Thr Asp Tyr Pro Leu
His Trp Val Lys Gln Ala Pro Gly Lys Gly 165 170 175Leu Gln Trp Met
Ala Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr 180 185 190Ala Asp
Asp Phe Thr Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala 195 200
205Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala
210 215 220Thr Tyr Phe Cys Val Arg Gly Tyr Tyr Tyr Tyr Trp Gly Gln
Gly Thr225 230 235 240Thr Leu Ser Val Ser Ser Asp Tyr Lys Asp Asp
Asp Asp Lys 245 25015765DNAArtificial SequenceRecombinantly
produced polynucleotide encoding single chain antibody of SEQ ID
NO14 and STOP codon 15atggacattg tgatgtcaca gtctccatcc tccctggctg
tgtcagcagg agagaaggtc 60actatgagct gcaaatccag tcagagtctg ctcaacagta
gaacccgaaa gaataacttg 120gcttggtacc agcagaaacc agggcagtct
cctaaactgc tgatctactg ggcatccact 180agggaatctg gggtccctga
tcgcttcaca ggcagtggat ctgggacaga tttcactctc 240accatcagca
gtgtgcaggc tgaagacctg gcagtttatt actgcaagca atcttataat
300ctgtggacgt tcggtggagg caccaagctg gaaatcaaag tcgacggtgg
tggtggttct 360ggcggcggcg gctccggtgg tggtggttct ctcgagcaga
tccagttggt gcagtctgga 420cctgagctga agaagcctgg agagacagtc
aagatctcct gcaaggcttc tggttatacc 480ttcacagact atccattgca
ctgggtgaag caggctccag gaaagggttt acagtggatg 540gcctggataa
acactgagac tggtgagcca acatatgcag atgacttcac gggacggttt
600gccttctctt tggagacctc tgccagcact gcctatttgc agatcaacaa
cctcaaaaat 660gaggacacgg ctacatattt ctgtgttaga ggttattatt
actactgggg ccaaggcacc 720actctctcag tctcctcaga ctacaaagac
gatgacgata aatag 76516259PRTArtificial SequenceRecombinantly
produced single chain antibody 16Gln Ala Gln Ile Gln Leu Val Gln
Ser Gly Pro Glu Leu Lys Lys Pro1 5 10 15Gly Glu Thr Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30Asp Tyr Pro Leu His Trp
Val Lys Gln Ala Pro Gly Lys Gly Leu Gln 35 40 45Trp Met Ala Trp Ile
Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp 50 55 60Asp Phe Thr Gly
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr65 70 75 80Ala Tyr
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr 85 90 95Phe
Cys Val Arg Gly Tyr Tyr Tyr Tyr Trp Gly Gln Gly Thr Thr Leu 100 105
110Ser Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly 130 135 140Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser Ser
Leu Ala Val Ser145 150 155 160Ala Gly Glu Lys Val Thr Met Ser Cys
Lys Ser Ser Gln Ser Leu Leu 165 170 175Asn Ser Arg Thr Arg Lys Asn
Asn Leu Ala Trp Tyr Gln Gln Lys Pro 180 185 190Gly Gln Ser Pro Lys
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser 195 200 205Gly Val Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 210 215 220Leu
Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys225 230
235 240Lys Gln Ser Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu
Glu 245 250 255Ile Lys Arg17777DNAArtificial SequenceRecombinantly
produced polynucleotide encoding single chain antibody of SEQ ID
NO16 17caagcacaga tccagttggt gcagtctgga cctgagctga agaagcctgg
agagacagtc 60aagatctcct gcaaggcttc tggttatacc ttcacagact atccattgca
ctgggtgaag 120caggctccag gaaagggttt acagtggatg gcctggataa
acactgagac tggtgagcca 180acatatgcag atgacttcac gggacggttt
gccttctctt tggagacctc tgccagcact 240gcctatttgc agatcaacaa
cctcaaaaat gaggacacgg ctacatattt ctgtgttaga 300ggttattatt
actactgggg ccaaggcacc actctctcag tctcctcagg tggtggtggt
360tctggcggcg gcggctccgg tggtggtggt agcggcggtg gtggttccgg
cggcggcggc 420tctggtggtg gtggttctga cattgtgatg tcacagtctc
catcctccct ggctgtgtca 480gcaggagaga aggtcactat gagctgcaaa
tccagtcaga gtctgctcaa cagtagaacc 540cgaaagaata acttggcttg
gtaccagcag aaaccagggc agtctcctaa actgctgatc 600tactgggcat
ccactaggga atctggggtc cctgatcgct tcacaggcag tggatctggg
660acagatttca ctctcaccat cagcagtgtg caggctgaag acctggcagt
ttattactgc 720aagcaatctt ataatctgtg gacgttcggt ggaggcacca
agctggaaat caaacgt 77718263PRTArtificial SequenceRecombinantly
produced single chain antibody with C-terminal His tag 18Gln Ile
Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Pro Leu His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Gln Trp Met
35 40 45Ala Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp
Phe 50 55 60Thr Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr
Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95Val Arg Gly Tyr Tyr Tyr Tyr Trp Gly Gln Gly
Thr Thr Leu Ser Val 100 105 110Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Asp Ile Val Met Ser
Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly145 150 155 160Glu Lys
Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 165 170
175Arg Thr Arg Lys Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
180 185 190Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 195 200 205Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 210 215 220Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
Val Tyr Tyr Cys Lys Gln225 230 235 240Ser Tyr Asn Leu Trp Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 245 250 255Arg His His His His
His His 26019831DNAArtificial SequenceRecombinantly produced
construct encoding single chain antibody with restriction sites and
His tag 19taatacgtac aagcacagat ccagttggtg cagtctggac ctgagctgaa
gaagcctgga 60gagacagtca agatctcctg caaggcttct ggttatacct tcacagacta
tccattgcac 120tgggtgaagc aggctccagg aaagggttta cagtggatgg
cctggataaa cactgagact 180ggtgagccaa catatgcaga tgacttcacg
ggacggtttg ccttctcttt ggagacctct 240gccagcactg cctatttgca
gatcaacaac ctcaaaaatg aggacacggc tacatatttc 300tgtgttagag
gttattatta ctactggggc caaggcacca ctctctcagt ctcctcaggt
360ggtggtggtt ctggcggcgg cggctccggt ggtggtggta gcggcggtgg
tggttccggc 420ggcggcggct ctggtggtgg tggttctgac attgtgatgt
cacagtctcc atcctccctg 480gctgtgtcag caggagagaa ggtcactatg
agctgcaaat ccagtcagag tctgctcaac 540agtagaaccc gaaagaataa
cttggcttgg taccagcaga aaccagggca gtctcctaaa 600ctgctgatct
actgggcatc cactagggaa tctggggtcc ctgatcgctt cacaggcagt
660ggatctggga cagatttcac tctcaccatc agcagtgtgc aggctgaaga
cctggcagtt 720tattactgca agcaatctta taatctgtgg acgttcggtg
gaggcaccaa gctggaaatc 780aaacgtcatc atcatcatca tcatggaggt
agttcttagg cggccgcata a 83120431DNAArtificial SequenceRecombinantly
produced construct encoding mouse antibody heavy chain variable
region, with restriction sites 20cagaattcgt gatctagtcg acatgggttg
ggtgtggaac ttgctattcc tgatggcagc 60tgcccaatgt atccaagcac agatccagtt
ggtgcagtct ggacctgagc tgaagaagcc 120tggagagaca gtcaagatct
cctgcaaggc ttctggttat accttcacag actatccatt 180gcactgggtg
aagcaggctc caggaaaggg tttacagtgg atggcctgga taaacactga
240gactggtgag ccaacatatg cagatgactt cacgggacgg tttgccttct
ctttggagac 300ctctgccagc actgcctatt tgcagatcaa caacctcaaa
aatgaggaca cggctacata 360tttctgtgtt agaggttatt attactactg
gggccaaggc accactctct cagtctcctc 420agctagcgta t
43121459DNAArtificial SequenceRecombinantly produced construct
encoding mouse antibody light chain variable
region, with restriction sites 21cacgaccggt ctagagctag cctaggctcg
agaagcttgt cgacgaattc agatactagt 60cgacatggtt ctcatgttac tgctgctatg
ggtatctggt acctgtgggg acattgtgat 120gtcacagtct ccatcctccc
tggctgtgtc agcaggagag aaggtcacta tgagctgcaa 180atccagtcag
agtctgctca acagtagaac ccgaaagaat aacttggctt ggtaccagca
240gaaaccaggg cagtctccta aactgctgat ctactgggca tccactaggg
aatctggggt 300ccctgatcgc ttcacaggca gtggatctgg gacagatttc
actctcacca tcagcagtgt 360gcaggctgaa gacctggcag tttattactg
caagcaatct tataatctgt ggacgttcgg 420tggaggcacc aagctggaaa
tcaaacgtac ggatgctgc 45922339DNAArtificial SequenceRecombinantly
produced polynucleotide encoding mouse antibody light chain
variable region 22gacattgtga tgtcacagtc tccatcctcc ctggctgtgt
cagcaggaga gaaggtcact 60atgagctgca aatccagtca gagtctgctc aacagtagaa
cccgaaagaa taacttggct 120tggtaccagc agaaaccagg gcagtctcct
aaactgctga tctactgggc atccactagg 180gaatctgggg tccctgatcg
cttcacaggc agtggatctg ggacagattt cactctcacc 240atcagcagtg
tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg
300tggacgttcg gtggaggcac caagctggaa atcaaacgg 33923342DNAArtificial
SequenceRecombinantly produced polynucleotide encoding mouse
antibody heavy chain variable region 23cagatccagt tggtgcagtc
tggacctgag ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg cttctggtta
taccttcaca gactatccat tgcactgggt gaagcaggct 120ccaggaaagg
gtttacagtg gatggcctgg ataaacactg agactggtga gccaacatat
180gcagatgact tcacgggacg gtttgccttc tctttggaga cctctgccag
cactgcctat 240ttgcagatca acaacctcaa aaatgaggac acggctacat
atttctgtgt tagaggttat 300tattactact ggggccaagg caccactctc
tcagtctcct ca 34224384DNAArtificial SequenceRecombinantly produced
polynucleotide encoding mouse antibody light chain variable region,
with signal sequence 24atggttctca tgttactgct gctatgggta tctggtacct
gtggggacat tgtgatgtca 60cagtctccat cctccctggc tgtgtcagca ggagagaagg
tcactatgag ctgcaaatcc 120agtcagagtc tgctcaacag tagaacccga
aagaataact tggcttggta ccagcagaaa 180ccagggcagt ctcctaaact
gctgatctac tgggcatcca ctagggaatc tggggtccct 240gatcgcttca
caggcagtgg atctgggaca gatttcactc tcaccatcag cagtgtgcag
300gctgaagacc tggcagttta ttactgcaag caatcttata atctgtggac
gttcggtgga 360ggcaccaagc tggaaatcaa acgg 38425399DNAArtificial
SequenceRecombinantly produced polynucleotide encoding mouse
antibody heavy chain variable region, with signal sequence
25atgggttggg tgtggaactt gctattcctg atggcagctg cccaatgtat ccaagcacag
60atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc
120tgcaaggctt ctggttatac cttcacagac tatccattgc actgggtgaa
gcaggctcca 180ggaaagggtt tacagtggat ggcctggata aacactgaga
ctggtgagcc aacatatgca 240gatgacttca cgggacggtt tgccttctct
ttggagacct ctgccagcac tgcctatttg 300cagatcaaca acctcaaaaa
tgaggacacg gctacatatt tctgtgttag aggttattat 360tactactggg
gccaaggcac cactctctca gtctcctca 39926113PRTArtificial
SequenceRecombinantly produced antibody light chain variable region
derived from mouse with native signal sequence removed and native
C-terminal arginine residue removed; including an inserted
N-terminal methionine residue 26Met Asp Ile Val Met Ser Gln Ser Pro
Ser Ser Leu Ala Val Ser Ala1 5 10 15Gly Glu Lys Val Thr Met Ser Cys
Lys Ser Ser Gln Ser Leu Leu Asn 20 25 30Ser Arg Thr Arg Lys Asn Asn
Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45Gln Ser Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 50 55 60Val Pro Asp Arg Phe
Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu65 70 75 80Thr Ile Ser
Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys 85 90 95Gln Ser
Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys27336DNAArtificial SequenceRecombinantly produced
polynucleotide encoding mouse antibody light chain variable region,
without native signal sequence and without native C-terminal
arginine residue 27gacattgtga tgtcacagtc tccatcctcc ctggctgtgt
cagcaggaga gaaggtcact 60atgagctgca aatccagtca gagtctgctc aacagtagaa
cccgaaagaa taacttggct 120tggtaccagc agaaaccagg gcagtctcct
aaactgctga tctactgggc atccactagg 180gaatctgggg tccctgatcg
cttcacaggc agtggatctg ggacagattt cactctcacc 240atcagcagtg
tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg
300tggacgttcg gtggaggcac caagctggaa atcaaa 33628339DNAArtificial
SequenceRecombinantly produced polynucleotide encoding mouse
antibody light chain variable region, without native signal
sequence and without native C-terminal arginine residue; including
an inserted N-terminal methionine residue 28atggacattg tgatgtcaca
gtctccatcc tccctggctg tgtcagcagg agagaaggtc 60actatgagct gcaaatccag
tcagagtctg ctcaacagta gaacccgaaa gaataacttg 120gcttggtacc
agcagaaacc agggcagtct cctaaactgc tgatctactg ggcatccact
180agggaatctg gggtccctga tcgcttcaca ggcagtggat ctgggacaga
tttcactctc 240accatcagca gtgtgcaggc tgaagacctg gcagtttatt
actgcaagca atcttataat 300ctgtggacgt tcggtggagg caccaagctg gaaatcaaa
339295PRTArtificial SequenceSynthesized linker segment 29Gly Gly
Gly Gly Ser1 530831DNAArtificial SequenceRecombinantly produced
construct encoding single chain antibody with restriction sites and
His Tag; complementary strand to SEQ ID NO19 30ttatgcggcc
gcctaagaac tacctccatg atgatgatga tgatgacgtt tgatttccag 60cttggtgcct
ccaccgaacg tccacagatt ataagattgc ttgcagtaat aaactgccag
120gtcttcagcc tgcacactgc tgatggtgag agtgaaatct gtcccagatc
cactgcctgt 180gaagcgatca gggaccccag attccctagt ggatgcccag
tagatcagca gtttaggaga 240ctgccctggt ttctgctggt accaagccaa
gttattcttt cgggttctac tgttgagcag 300actctgactg gatttgcagc
tcatagtgac cttctctcct gctgacacag ccagggagga 360tggagactgt
gacatcacaa tgtcagaacc accaccacca gagccgccgc cgccggaacc
420accaccgccg ctaccaccac caccggagcc gccgccgcca gaaccaccac
cacctgagga 480gactgagaga gtggtgcctt ggccccagta gtaataataa
cctctaacac agaaatatgt 540agccgtgtcc tcatttttga ggttgttgat
ctgcaaatag gcagtgctgg cagaggtctc 600caaagagaag gcaaaccgtc
ccgtgaagtc atctgcatat gttggctcac cagtctcagt 660gtttatccag
gccatccact gtaaaccctt tcctggagcc tgcttcaccc agtgcaatgg
720atagtctgtg aaggtataac cagaagcctt gcaggagatc ttgactgtct
ctccaggctt 780cttcagctca ggtccagact gcaccaactg gatctgtgct
tgtacgtatt a 831
* * * * *