U.S. patent application number 14/997849 was filed with the patent office on 2017-06-01 for anti-inflammatory molecules with tissue-targeting functions.
This patent application is currently assigned to Immunwork Inc.. The applicant listed for this patent is Immunwork Inc.. Invention is credited to Tse-Wen Chang, Jou-Han Chen, Hsing-Mao Chu.
Application Number | 20170152323 14/997849 |
Document ID | / |
Family ID | 56405263 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170152323 |
Kind Code |
A1 |
Chang; Tse-Wen ; et
al. |
June 1, 2017 |
Anti-inflammatory molecules with tissue-targeting functions
Abstract
The present disclosure provides various molecular constructs
having a targeting element and an effector element. Methods for
treating various diseases using such molecular constructs are also
disclosed.
Inventors: |
Chang; Tse-Wen; (Taipei,
TW) ; Chen; Jou-Han; (Taipei, TW) ; Chu;
Hsing-Mao; (Taipei, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunwork Inc. |
Taipei |
|
TW |
|
|
Assignee: |
Immunwork Inc.
Taipei
TW
|
Family ID: |
56405263 |
Appl. No.: |
14/997849 |
Filed: |
January 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62104405 |
Jan 16, 2015 |
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62114427 |
Feb 10, 2015 |
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62137737 |
Mar 24, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6883 20170801;
C07K 16/32 20130101; A61K 31/739 20130101; A61P 29/00 20180101;
A61P 35/02 20180101; C07K 14/655 20130101; A61P 27/02 20180101;
C07K 14/705 20130101; C07K 2319/30 20130101; C07K 2319/32 20130101;
A61P 1/04 20180101; A61K 47/58 20170801; A61K 47/60 20170801; A61K
47/6803 20170801; C07K 14/70578 20130101; C07K 2317/732 20130101;
A61K 47/61 20170801; C07K 2317/71 20130101; A61K 47/6849 20170801;
A61K 51/088 20130101; C07K 16/241 20130101; C07K 2317/31 20130101;
C07K 2317/24 20130101; C07K 2317/526 20130101; A61K 47/64 20170801;
C07K 16/244 20130101; C07K 2317/73 20130101; A61K 2039/505
20130101; A61P 37/06 20180101; C07K 2317/55 20130101; A61P 37/00
20180101; C07K 16/468 20130101; C07K 2317/524 20130101; A61K 31/397
20130101; C07K 2319/33 20130101; A61P 19/10 20180101; C07K 14/485
20130101; C07K 2317/94 20130101; A61K 47/6851 20170801; C07K
16/2887 20130101; C07K 16/2875 20130101; C07K 16/18 20130101; C07K
16/2863 20130101; C07K 2317/76 20130101; A61P 17/06 20180101; C07K
16/2803 20130101; A61K 47/6801 20170801; A61K 51/065 20130101; A61P
37/02 20180101; A61K 31/4709 20130101; C07K 2317/64 20130101; A61K
31/4545 20130101; A61P 19/02 20180101; A61P 35/00 20180101; C07K
14/7151 20130101; C07K 16/22 20130101; C07K 2317/622 20130101; A61K
31/537 20130101; A61K 47/6843 20170801; C07K 2317/21 20130101; A61K
31/4745 20130101; C07K 16/2809 20130101; A61K 47/6845 20170801;
C07K 16/2818 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/28 20060101 C07K016/28; C07K 14/705 20060101
C07K014/705; C07K 16/18 20060101 C07K016/18; C07K 16/24 20060101
C07K016/24 |
Claims
1. A molecular construct comprising, a pair of CH2-CH3 segments of
an IgG.Fc; a first pair of effector elements, wherein the effector
element is an antibody fragment specific for tumor necrosis
factor-.alpha. (TNF-.alpha.), interleukin-17 (IL-17), IL-17
receptor (IL-17R), IL-1, IL-6, IL-6R, IL-12, IL-23, B cell
activating factor (BAFF), or receptor activator of nuclear factor
kappa-B ligand (RANKL); or a soluble receptor of TNF-.alpha. or
IL-1; and a first pair of targeting elements, wherein the targeting
element is an antibody fragment specific for .alpha.-aggrecan,
collagen I, collagen II, collagen III, collagen V, collagen VII,
collagen IX, collagen XI, or osteonectin, wherein, when the first
pair of effector elements is linked to the N-termini of the pair of
CH2-CH3 segments, then the first pair of targeting elements is
linked to the C-termini of the pair of CH2-CH3 segments, and vice
versa, or when the first pair of effectors elements and the first
pair of targeting elements are both in the form of single-chain
variable fragments (scFvs), then the first pair of targeting
elements is linked to the N-termini of the first pair of effector
elements in a tandem or diabody configuration, thereby forming a
pair of bispecific scFvs that are linked to the N-termini of the
pair of CH2-CH3 segments.
2. The molecular construct of claim 1, wherein the pair of CH2-CH3
segments is derived from human .gamma.4 or .gamma.1
immunoglobulin.
3. The molecular construct of claim 1, wherein when the first pair
of effector elements is in the form of an antigen-binding fragment
(Fab), and the first pair of targeting elements is in the form of
scFvs, and vice versa; then the Fab and scFvs are respectively
linked to the N-termini and C-termini of the CH2-CH3 segments, so
that molecular construct adopts an extended IgG configuration.
4. The molecular construct of claim 1, further comprising a second
pair of effector elements or a second pair of targeting elements,
wherein the second pair of effector or targeting elements is linked
to the free C-termini of the CH2-CH3 segments.
5. The molecular construct of claim 1, wherein, the effector
element is an scFv specific for TNF-.alpha.; and the targeting
element is an scFv specific for collagen II, collagen IX, or
.alpha.-aggrecan.
6. The molecular construct of claim 1, wherein, the two effector
elements are in the form of a Fab antibody specific for
TNF-.alpha.; and the targeting element is an scFv specific for
collagen II or collagen IX.
7. The molecular construct of claim 1, wherein, the effector
element is an scFv specific for IL-17; and the targeting element is
an scFv specific for collagen I or collagen VII.
8. The molecular construct of claim 1, wherein, the two effector
elements are in the form of a Fab antibody specific for IL-17; and
the targeting element is an scFv specific for collagen I or
collagen VII.
9. The molecular construct of claim 1, wherein, the effector
element is an scFv specific for BAFF; and the targeting element is
an scFv specific for collagen I or collagen VII.
10. The molecular construct of claim 1, wherein, the two effector
elements are in the form of a Fab antibody specific for BAFF; and
the targeting element is an scFv specific for collagen I or
collagen VII.
11. The molecular construct of claim 1, wherein, the effector
element is an scFv specific for TNF-.alpha.; and the targeting
element is an scFv specific for collagen III or collagen V.
12. The molecular construct of claim 1, wherein, the two effector
elements are in the form of a Fab antibody specific for
TNF-.alpha.; and the targeting element is an scFv specific for
collagen III or collagen V.
13. The molecular construct of claim 1, wherein, the effector
element is an scFv specific for RANKL; and the targeting element is
an scFv specific for collagen I or osteonectin.
14. The molecular construct of claim 1, wherein, the two effector
elements are in the form of a Fab specific for RANKL; and the
targeting element is an scFv specific for collagen I or
osteonectin.
15. A method for treating an immune disorder, comprising the step
of administering to a subject in need thereof an effective amount
of the molecular construct according to claim 1.
16. The method of claim 15, wherein the immune disorder is an
autoimmune disease.
17. The method of claim 16, wherein, the autoimmune disease is
rheumatoid arthritis, psoriatic arthritis, or ankylosing
spondylitis; the effector element is an antibody fragment specific
for TNF-.alpha., IL-12/IL-23, IL-1, IL-17, or IL-6; and the
targeting element is an antibody fragment specific for collagen II,
collagen IX, collagen XI, or .alpha.-aggrecan.
18. The method of claim 16, wherein, the autoimmune disease is
psoriasis; the effector element is an antibody fragment specific
for TNF-.alpha., IL-12/IL-23, or IL-17; and the targeting element
is an antibody fragment specific for collagen I or collagen
VII.
19. The method of claim 16, wherein, the autoimmune disease is
systemic lupus erythematosus, cutaneous lupus, or Sjogren's
Syndrome; the effector element is an antibody fragment specific for
BAFF; and the targeting element is an antibody fragment specific
for collagen I or collagen VII.
20. The method of claim 16, wherein, the autoimmune disease is an
inflammatory bowel disease; the effector element is an antibody
fragment specific for TNF-.alpha.; and the targeting element is an
antibody fragment specific for collagen III or collagen V.
21. The method of claim 20, wherein the inflammatory bowel disease
is Crohn's disease or ulcerative colitis.
22. A method for treating osteoporosis, comprising the step of
administering to a subject in need thereof an effective amount of
the molecular construct according to claim 1.
23. The method of claim 22, wherein, of the effector element is an
antibody fragment specific for RANKL; and the targeting element is
an antibody fragment specific for collagen I or osteonectin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application relates to and claims the benefit of U.S.
Provisional Application No. 62/104,405, filed Jan. 16, 2015, U.S.
Provisional Application No. 62/114,427, filed Feb. 10, 2015, and
U.S. Provisional Application No. 62/137,737, filed Mar. 24, 2015;
the contents of the applications are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present disclosure relates to the field of
pharmaceuticals; more particularly, to multi-functional molecular
constructs, e.g., those having targeting and effector elements for
delivering the effector (e.g., therapeutic drug) to targeted
sites.
[0004] 2. Description of the Related Art
[0005] The continual advancement of a broad array of methodologies
for screening and selecting monoclonal antibodies (mAbs) for
targeted antigens has helped the development of a good number of
therapeutic antibodies for many diseases that were regarded as
untreatable just a few years ago. According to Therapeutic Antibody
Database, approximately 2,800 antibodies have been studied or are
being planned for studies in human clinical trials, and
approximately 80 antibodies have been approved by governmental drug
regulatory agencies for clinical uses. The large amount of data on
the therapeutic effects of antibodies has provided information
concerning the pharmacological mechanisms how antibodies act as
therapeutics.
[0006] One major pharmacologic mechanism for antibodies acting as
therapeutics is that, antibodies can neutralize or trap
disease-causing mediators, which may be cytokines or immune
components present in the blood circulation, interstitial space, or
in the lymph nodes. The neutralizing activity inhibits the
interaction of the disease-causing mediators with their receptors.
It should be noted that fusion proteins of the soluble receptors or
the extracellular portions of receptors of cytokines and the Fc
portion of IgG, which act by neutralizing the cytokines or immune
factors in a similar fashion as neutralizing antibodies, have also
been developed as therapeutic agents.
[0007] Several therapeutic antibodies that have been approved for
clinical applications or subjected to clinical developments mediate
their pharmacologic effects by binding to receptors, thereby
blocking the interaction of the receptors with their ligands. For
those antibody drugs, Fc-mediated mechanisms, such as
antibody-dependent cellular cytotoxicity (ADCC) and
complement-mediated cytolysis (CMC), are not the intended
mechanisms for the antibodies.
[0008] Some therapeutic antibodies bind to certain surface antigens
on target cells and render Fc-mediated functions and other
mechanisms on the target cells. The most important Fc-mediated
mechanisms are antibody-dependent cellular cytotoxicity (ADCC) and
complement-mediated cytolysis (CMC), which both will cause the
lysis of the antibody-bound target cells. Some antibodies binding
to certain cell surface antigens can induce apoptosis of the bound
target cells.
[0009] Antibodies can also serve as carriers of cytotoxic molecules
or other therapeutic agents without the antibodies' serving obvious
therapeutic effector functions. In general, those antibodies bind
to "tumor-associated" antigens on target cells, but cannot cause
cell lysis by themselves. Antibodies specific for CD19 and CD22 on
B lymphomas are well known. For many years, those antibodies have
been explored as carriers for cytotoxic agents, including
radioactive nuclides with very short half-lives, such as .sup.90Y,
.sup.131I, and .sup.177Lu. Some antibodies have also been studied
as targeting agents for liposomes loaded with cytotoxic drugs, such
as doxorubicin, paclitaxel, and amphotericin B. The field of
antibody drug conjugates (ADC) has experienced an explosive phase
of research and development in recent years, mainly attributing to
the development of extremely cytotoxic drugs, such as auristatin,
maytansine, calicheamicin, and camptothecin, and of methodologies
for conjugating the cytotoxic molecules onto antibody molecules.
Those ADCs have been designed to target diffusive (or liquid)
tumors of the blood, lymphoid system, and bone marrow, including
various types of lymphomas and leukemia, expressing one or more
unique CD markers. Some ADCs are also being developed for solid
tumors. A few of this new generation of antibody drug conjugates
have been approved for clinical uses and many are in clinical
trials.
[0010] However, in the first generation of ADCs, the cytotoxic drug
molecules are linked non-selectively to cysteine or lysine residues
in the antibody, thereby resulting in a heterogeneous mixture of
ADCs with different numbers of drug molecules per ADC. This
approach leads to some safety and efficacy issues. For example, the
first FDA-approved ADC, gemtuzumab ozogamicin, for treating acute
myelogenous leukemia, is now withdrawn from the market due to
unacceptable toxicity.
[0011] The concept and methodology for preparing antibodies with
dual specificities germinated more than three decades ago. In
recent year, the advancement in recombinant antibody engineering
methodologies and the drive to develop improved medicine has
stimulated the development bi-specific antibodies adopting a large
variety of structural configurations.
[0012] For example, the bi-valent or multivalent antibodies may
contain two or more antigen-binding sites. A number of methods have
been reported for preparing multivalent antibodies by covalently
linking three or four Fab fragments via a connecting structure. For
example, antibodies have been engineered to express tandem three or
four Fab repeats.
[0013] Several methods for producing multivalent antibodies by
employing synthetic crosslinkers to associate, chemically,
different antibodies or binding fragments have been disclosed. One
approach involves chemically cross-linking three, four, and more
separately Fab fragments using different linkers. Another method to
produce a construct with multiple Fabs that are assembled to
one-dimensional DNA scaffold was provided.
[0014] Those various multivalent Ab constructs designed for binding
to target molecules differ among one another in size, half-lives,
flexibility in conformation, and ability to modulate the immune
system. In view of the foregoing, several reports have been made
for preparing molecular constructs with a fixed number of effector
elements or with two or more different kinds of functional elements
(e.g., at least one targeting element and at least one effector
element). However, it is often difficult to build a molecular
construct with a particular combination of the targeting and
effector elements either using chemical synthesis or recombinant
technology. Accordingly, there exists a need in the related art to
provide novel molecular platforms to build a more versatile
molecule suitable for covering applications in a wide range of
diseases.
SUMMARY
[0015] The following presents a simplified summary of the
disclosure in order to provide a basic understanding to the reader.
This summary is not an extensive overview of the disclosure and it
does not identify key/critical elements of the present invention or
delineate the scope of the present invention. Its sole purpose is
to present some concepts disclosed herein in a simplified form as a
prelude to the more detailed description that is presented
later.
[0016] In a first aspect, the present disclosure is directed to a
fragment crystallizable (Fc)-based molecular construct that has at
least one targeting element and at least one effector element
linked, directly or indirectly, to a CH2-CH3 domain of an
immunoglobulin. The design of the present Fc-based molecular
construct allows for numerous combinations of a wide range of
targeting and effector elements. Hence, the present Fc-based
molecular construct may serve as a platform for constructing
multi-valent molecules.
[0017] According to certain embodiments of the present disclosure,
the Fc-based molecular construct comprises a pair of CH2-CH3
segments of an IgG.Fc, a first pair of effector elements, and a
first pair of targeting elements.
[0018] In some embodiments, the present Fc-based molecular
constructs are intended to be used in the treatment of immune
diseases (in particular, autoimmune diseases) or osteoporosis. In
this case, the first pair of effector elements consists of two
effector elements, in which each of the two effector elements is an
antibody fragment specific for tumor necrosis factor-.alpha.
(TNF-.alpha.), interleukin-17 (IL-17), IL-17 receptor (IL-17R),
IL-1, IL-6, IL-6R, IL-12/IL-23, B cell activating factor (BAFF), or
a receptor activator of nuclear factor kappa-B ligand (RANKL); or a
soluble receptor of TNF-.alpha. or IL-1. Further, the first pair of
targeting elements consists of two targeting elements, in which
each of the two targeting elements is an antibody fragment specific
for .alpha.-aggrecan, collagen I, collagen II, collagen III,
collagen V, collagen VII, collagen IX, collagen XI, or osteonectin.
In the case where the first pair of effector elements is linked to
the N-termini of the pair of CH2-CH3 segments, the first pair of
targeting elements is linked to the C-termini of the pair of
CH2-CH3 segments, and vice versa. Alternatively, when the first
pair of effectors elements and the first pair of targeting elements
is both in the form of single-chain variable fragments (scFvs),
then the first pair of targeting elements is linked to the
N-termini of the first pair of effector elements in a tandem or
diabody configuration, thereby forming a pair of bispecific scFvs
that are linked to the N-termini of the pair of CH2-CH3
segments.
[0019] In certain embodiments, the pair of CH2-CH3 segments is
derived from human IgG heavy chain .gamma.4 or human IgG heavy
chain .gamma.1.
[0020] In some examples, the first pair of effector elements or the
first pair of the targeting elements takes a Fab configuration
(i.e., consisting of the V.sub.H-CH1 domain and the
V.sub.L-C.kappa. domain); this Fab fragment is linked to the
N-termini of the first and second heavy chains, so that the
Fc-based molecular construct adopts an IgG configuration. In these
cases, the pair of elements that is not in the Fab configuration is
linked to the C-termini of the pair of CH2-CH3 segments.
[0021] According to other embodiments, the Fc-based molecular
construct further comprises a second pair of effector elements,
which consists of two additional effector elements that are both
selected from the group described above for the effector elements.
According to various embodiments, the elements of the second pair
of effector elements are different from those of the first pair of
effector elements. In these embodiments, the second pair of
effector elements is linked to the free C-termini of the CH2-CH3
segments.
[0022] Alternatively, the present Fc-based molecular construct
further comprises a second pair of targeting elements, in which the
two targeting elements are both selected from the group described
above regarding the targeting elements. According to various
embodiments, the elements of the second pair of targeting elements
are different from those of the first pair of targeting elements.
In these embodiments, the second pair of targeting elements is
linked to the free C-termini of the CH2-CH3 segments.
[0023] According to various optional embodiments, the targeting
elements and effector elements described above can be combined as
desired, so as to attain the intended therapeutic effect. Some
exemplary combination of the effector element(s) and targeting
element(s) for treating immune diseases are provided in the
appended claims and discussed in the description section
bellow.
[0024] In a second aspect, the present disclosure is directed to
methods for treating various diseases. Generally, the methods
involve the step of administrating an effective amount of the
Fc-based molecular constructs according to the first aspect and any
of the associated embodiments, to a subject in need of such
treatment.
[0025] In certain embodiments, the present method is directed to
the treatment of an immune disease; in particular, an autoimmune
disease.
[0026] According to some embodiments of the present disclosure, the
autoimmune disease is rheumatoid arthritis, psoriatic arthritis, or
ankylosing spondylitis. In this case, the effector element is an
antibody fragment specific for TNF-.alpha., IL-12/IL-23, IL-1,
IL-17, or IL-6, while the targeting element may be an antibody
fragment specific for collagen II, collagen IX, collagen XI, or
.alpha.-aggrecan.
[0027] According to various embodiments, the autoimmune disease is
psoriasis. In this case, the effector element is an antibody
fragment specific for TNF-.alpha., IL-12/IL-23, or IL-17, while the
targeting element is an antibody fragment specific for collagen I
or collagen VII.
[0028] According to some other embodiments, the autoimmune disease
is systemic lupus erythematosus, cutaneous lupus, or Sjogren's
Syndrome. In this case, the effector element is an antibody
fragment specific for BAFF, and the targeting element is an
antibody fragment specific for collagen I, or collagen VII.
[0029] According to some embodiments, the autoimmune disease is an
inflammatory bowel disease, such as Crohn's disease or ulcerative
colitis. In this case, the effector element is an antibody fragment
specific for TNF-.alpha., and the targeting element is an antibody
fragment specific for collagen III or collagen V.
[0030] Another disease treatable by the method proposed herein is
osteoporosis. According to embodiments of the present disclosure,
the effector element for treating osteoporosis comprises an
antibody fragment specific for RANKL, while the targeting element
comprises an antibody fragment specific for collagen I or
osteonectin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The present description will be better understood from the
following detailed description read in light of the accompanying
drawings briefly discussed below.
[0032] FIGS. 1A to 1F are schematic diagrams illustrating Fc-based
molecular constructs according to various embodiments of the
present disclosure.
[0033] FIGS. 2A and 2B are schematic diagrams illustrating Fc-based
molecular constructs according to various embodiments of the
present disclosure.
[0034] FIG. 3 shows the SDS-PAGE analysis of (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc.
[0035] FIGS. 4A and 4B show the ELISA results analyzing the binding
of (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc to
collagen II and TNF-.alpha..
[0036] FIGS. 5A and 5B respectively show the SDS-PAGE and ELISA
analysis of the 2-chain (scFv .alpha. CII)-(scFv .alpha.
TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17).
[0037] FIGS. 6A and 6B respectively show the SDS-PAGE and ELISA
analysis of 2-chain (soluble TNF-.alpha.
receptor)-IgG1.CH2-CH3-scFv .alpha. collagen II.
[0038] FIG. 7 shows the SDS-PAGE analysis of the 2-chain fusion
protein containing intact antibody for human TNF-.alpha. and scFv
specific for collagen II.
[0039] FIGS. 8A and 8B respectively show the SDS-PAGE and ELISA
analyses of the 2-chain fusion protein containing intact antibody
for human IL-17 and scFv specific for collagen VII.
[0040] FIGS. 9A and 9B respectively show the SDS-PAGE and ELISA
analysis of scFv .alpha. collagen VII-IgG4.CH2-CH3-scFv .alpha.
BAFF.
[0041] FIGS. 10A and 10B respectively show the SDS-PAGE and ELISA
analyses of the 2-chain fusion protein containing intact antibody
for human BAFF and scFv specific for collagen VII.
[0042] FIGS. 11A and 11B respectively show the SDS-PAGE and ELISA
analyses of the 2-chain (scFv .alpha. SPARC)-(scFv .alpha.
RANKL)-hIgG4.Fc molecular construct.
[0043] FIGS. 12A and 12B respectively show the SDS-PAGE and ELISA
analyses of the 2-chain fusion protein containing intact antibody
for human RANKL and scFv specific for human osteonectin.
[0044] FIG. 13 shows the immunostaining of mouse epiphyseal bone
with (scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc and
2-chain (scFv .alpha. CII)-(scFv .alpha.
TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17).
[0045] FIG. 14 shows the immunostaining of mouse epiphyseal bone
with 2-chain (soluble TNF-.alpha. receptor)-IgG1.CH2-CH3-scFv
.alpha. collagen II.
[0046] FIG. 15 shows bio-distribution of fluorescence-labeled (scFv
.alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc in vivo in BALB/c
mice.
[0047] In accordance with common practice, the various described
features/elements are not drawn to scale but instead are drawn to
best illustrate specific features/elements relevant to the present
invention. Also, like reference numerals and designations in the
various drawings are used to indicate like elements/parts, where
possible.
DESCRIPTION
[0048] The detailed description provided below in connection with
the appended drawings is intended as a description of the present
examples and is not intended to represent the only forms in which
the present example may be constructed or utilized. The description
sets forth the functions of the example and the sequence of steps
for constructing and operating the example. However, the same or
equivalent functions and sequences may be accomplished by different
examples.
[0049] For convenience, certain terms employed in the
specification, examples and appended claims are collected here.
Unless otherwise defined herein, scientific and technical
terminologies employed in the present disclosure shall have the
meanings that are commonly understood and used by one of ordinary
skill in the art.
[0050] Unless otherwise required by context, it will be understood
that singular terms shall include plural forms of the same and
plural terms shall include the singular. Specifically, as used
herein and in the claims, the singular forms "a" and "an" include
the plural reference unless the context clearly indicated
otherwise. Also, as used herein and in the claims, the terms "at
least one" and "one or more" have the same meaning and include one,
two, three, or more. Furthermore, the phrases "at least one of A,
B, and C", "at least one of A, B, or C" and "at least one of A, B
and/or C," as use throughout this specification and the appended
claims, are intended to cover A alone, B alone, C alone, A and B
together, B and C together, A and C together, as well as A, B, and
C together.
[0051] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in the respective testing measurements.
Also, as used herein, the term "about" generally means within 10%,
5%, 1%, or 0.5% of a given value or range. Alternatively, the term
"about" means within an acceptable standard error of the mean when
considered by one of ordinary skill in the art. Other than in the
operating/working examples, or unless otherwise expressly
specified, all of the numerical ranges, amounts, values and
percentages such as those for quantities of materials, durations of
times, temperatures, operating conditions, ratios of amounts, and
the likes thereof disclosed herein should be understood as modified
in all instances by the term "about." Accordingly, unless indicated
to the contrary, the numerical parameters set forth in the present
disclosure and attached claims are approximations that can vary as
desired. At the very least, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques. Ranges can be
expressed herein as from one endpoint to another endpoint or
between two endpoints. All ranges disclosed herein are inclusive of
the endpoints, unless specified otherwise.
[0052] This present disclosure pertains generally to molecular
constructs, in which each molecular construct comprises a targeting
element (T) and an effector element (E), and these molecular
constructs are sometimes referred to as "T-E molecules", "T-E
pharmaceuticals" or "T-E drugs" in this document.
[0053] As used herein, the term "targeting element" refers to the
portion of a molecular construct that directly or indirectly binds
to a target of interest (e.g., a receptor on a cell surface or a
protein in a tissue) thereby facilitates the transportation of the
present molecular construct into the interested target. In some
example, the targeting element may direct the molecular construct
to the proximity of the target cell. In other cases, the targeting
element specifically binds to a molecule present on the target cell
surface or to a second molecule that specifically binds a molecule
present on the cell surface. In some cases, the targeting element
may be internalized along with the present molecular construct once
it is bound to the interested target, hence is relocated into the
cytosol of the target cell. A targeting element may be an antibody
or a ligand for a cell surface receptor, or it may be a molecule
that binds such antibody or ligand, thereby indirectly targeting
the present molecular construct to the target site (e.g., the
surface of the cell of choice). The localization of the effector
(therapeutic agent) in the diseased site will be enhanced or
favored with the present molecular constructs as compared to the
therapeutic without a targeting function. The localization is a
matter of degree or relative proportion; it is not meant for
absolute or total localization of the effector to the diseased
site.
[0054] According to the present invention, the term "effector
element" refers to the portion of a molecular construct that
elicits a biological activity (e.g., inducing immune responses,
exerting cytotoxic effects and the like) or other functional
activity (e.g., recruiting other hapten tagged therapeutic
molecules), once the molecular construct is directed to its target
site. The "effect" can be therapeutic or diagnostic. The effector
elements encompass those that bind to cells and/or extracellular
immunoregulatory factors. The effector element comprises agents
such as proteins, nucleic acids, lipids, carbohydrates,
glycopeptides, drug moieties (both small molecule drug and
biologics), compounds, elements, and isotopes, and fragments
thereof.
[0055] Although the terms, first, second, third, etc., may be used
herein to describe various elements, components, regions, and/or
sections, these elements (as well as components, regions, and/or
sections) are not to be limited by these terms. Also, the use of
such ordinal numbers does not imply a sequence or order unless
clearly indicated by the context. Rather, these terms are simply
used to distinguish one element from another. Thus, a first
element, discussed below, could be termed a second element without
departing from the teachings of the exemplary embodiments.
[0056] Here, the terms "link," "couple," and "conjugates" are used
interchangeably to refer to any means of connecting two components
either via direct linkage or via indirect linkage between two
components.
[0057] The term "polypeptide" as used herein refers to a polymer
having at least two amino acid residues. Typically, the polypeptide
comprises amino acid residues ranging in length from 2 to about 200
residues; preferably, 2 to 50 residues. Where an amino acid
sequence is provided herein, L-, D-, or beta amino acid versions of
the sequence are also contemplated. Polypeptides also include amino
acid polymers in which one or more amino acid residues are an
artificial chemical analogue of a corresponding naturally occurring
amino acid, as well as to naturally occurring amino acid polymers.
In addition, the term applies to amino acids joined by a peptide
linkage or by other, "modified linkages" (e.g., where the peptide
bond is replaced by an .alpha.-ester, a .beta.-ester, a thioamide,
phosphonamide, carbomate, hydroxylate, and the like.
[0058] In certain embodiments, conservative substitutions of the
amino acids comprising any of the sequences described herein are
contemplated. In various embodiments, one, two, three, four, or
five different residues are substituted. The term "conservative
substitution" is used to reflect amino acid substitutions that do
not substantially alter the activity (e.g., biological or
functional activity and/or specificity) of the molecule. Typically,
conservative amino acid substitutions involve substitution one
amino acid for another amino acid with similar chemical properties
(e.g., charge or hydrophobicity). Certain conservative
substitutions include "analog substitutions" where a standard amino
acid is replaced by a non-standard (e.g., rare, synthetic, etc.)
amino acid differing minimally from the parental residue. Amino
acid analogs are considered to be derived synthetically from the
standard amino acids without sufficient change to the structure of
the parent, are isomers, or are metabolite precursors.
[0059] In certain embodiments, polypeptides comprising at least
80%, preferably at least 85% or 90%, and more preferably at least
95% or 98% sequence identity with any of the sequences described
herein are also contemplated.
[0060] "Percentage (%) amino acid sequence identity" with respect
to the polypeptide sequences identified herein is defined as the
percentage of polypeptide residues in a candidate sequence that are
identical with the amino acid residues in the specific polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percentage
sequence identity can be achieved in various ways that are within
the skill in the art, for instance, using publicly available
computer software such as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, sequence
comparison between two polypeptide sequences was carried out by
computer program Blastp (protein-protein BLAST) provided online by
Nation Center for Biotechnology Information (NCBI). The percentage
amino acid sequence identity of a given polypeptide sequence A to a
given polypeptide sequence B (which can alternatively be phrased as
a given polypeptide sequence A that has a certain % amino acid
sequence identity to a given polypeptide sequence B) is calculated
by the formula as follows:
X Y .times. 100 % ##EQU00001##
[0061] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program BLAST in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in A or B, whichever is shorter.
[0062] The term "PEGylated amino acid" as used herein refers to a
polyethylene glycol (PEG) chain with one amino group and one
carboxyl group. Generally, the PEGylated amino acid has the formula
of NH.sub.2--(CH.sub.2CH.sub.2O).sub.n--COOH. In the present
disclosure, the value of n ranges from 1 to 20; preferably, ranging
from 2 to 12.
[0063] As used herein, the term "terminus" with respect to a
polypeptide refers to an amino acid residue at the N- or C-end of
the polypeptide. With regard to a polymer, the term "terminus"
refers to a constitutional unit of the polymer (e.g., the
polyethylene glycol of the present disclosure) that is positioned
at the end of the polymeric backbone. In the present specification
and claims, the term "free terminus" is used to mean the terminal
amino acid residue or constitutional unit is not chemically bound
to any other molecular.
[0064] The term "antigen" or "Ag" as used herein is defined as a
molecule that elicits an immune response. This immune response may
involve a secretory, humoral and/or cellular antigen-specific
response. In the present disclosure, the term "antigen" can be a
protein, a polypeptide (including mutants or biologically active
fragments thereof), a polysaccharide, a glycoprotein, a glycolipid,
a nucleic acid, or a combination thereof.
[0065] In the present specification and claims, the term "antibody"
is used in the broadest sense and covers fully assembled
antibodies, antibody fragments that bind with antigens, such as
antigen-binding fragment (Fab/Fab'), F(ab').sub.2 fragment (having
two antigen-binding Fab portions linked together by disulfide
bonds), variable fragment (Fv), single chain variable fragment
(scFv), bi-specific single-chain variable fragment (bi-scFv),
nanobodies, unibodies and diabodies. "Antibody fragments" comprise
a portion of an intact antibody, preferably the antigen-binding
region or variable region of the intact antibody. Typically, an
"antibody" refers to a protein consisting of one or more
polypeptides substantially encoded by immunoglobulin genes or
fragments of immunoglobulin genes. The well-known immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon, and
mu constant region genes, as well as myriad immunoglobulin variable
region genes. Light chains are classified as either kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or
epsilon, which in turn define the immunoglobulin classes, IgG, IgM,
IgA, IgD, and IgE, respectively. A typical immunoglobulin
(antibody) structural unit is known to comprise a tetramer. Each
tetramer is composed of two identical pairs of polypeptide chains,
with each pair having one "light" chain (about 25 kDa) and one
"heavy" chain (about 50-70 kDa). The N-terminus of each chain
defines a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The terms variable
light chain (V.sub.L) and variable heavy chain (V.sub.H) refer to
these light and heavy chains, respectively. According to
embodiments of the present disclosure, the antibody fragment can be
produced by modifying the nature antibody or by de novo synthesis
using recombinant DNA methodologies. In certain embodiments of the
present disclosure, the antibody and/or antibody fragment can be
bispecific, and can be in various configurations. For example,
bispecific antibodies may comprise two different antigen binding
sites (variable regions). In various embodiments, bispecific
antibodies can be produced by hybridoma technique or recombinant
DNA technique. In certain embodiments, bispecific antibodies have
binding specificities for at least two different epitopes.
[0066] The term "specifically binds" as used herein, refers to the
ability of an antibody or an antigen-binding fragment thereof, to
bind to an antigen with a dissociation constant (Kd) of no more
than about 1.times.10.sup.-6 M, 1.times.10.sup.-7 M,
1.times.10.sup.-8 M, 1.times.10.sup.-9 M, 1.times.10.sup.-10 M,
1.times.10.sup.-11 M, 1.times.10.sup.-12 M, and/or to bind to an
antigen with an affinity that is at least two-folds greater than
its affinity to a nonspecific antigen.
[0067] The term "immune disorder" as used herein refers to a
disorder involving deficiency of humoral immunity, deficiency of
cell-mediated immunity, combined immunity deficiency, unspecified
immunity deficiency, and autoimmune disease.
[0068] The term "treatment" as used herein includes preventative
(e.g., prophylactic), curative or palliative treatment; and
"treating" as used herein also includes preventative (e.g.,
prophylactic), curative or palliative treatment. In particular, the
term "treating" as used herein refers to the application or
administration of the present molecular construct or a
pharmaceutical composition comprising the same to a subject, who
has a medical condition a symptom associated with the medical
condition, a disease or disorder secondary to the medical
condition, or a predisposition toward the medical condition, with
the purpose to partially or completely alleviate, ameliorate,
relieve, delay onset of, inhibit progression of, reduce severity
of, and/or reduce incidence of one or more symptoms or features of
said particular disease, disorder, and/or condition. Treatment may
be administered to a subject who does not exhibit signs of a
disease, disorder, and/or condition, and/or to a subject who
exhibits only early signs of a disease, disorder and/or condition,
for the purpose of decreasing the risk of developing pathology
associated with the disease, disorder and/or condition.
[0069] The term "effective amount" as used herein refers to the
quantity of the present recombinant protein that is sufficient to
yield a desired therapeutic response. An effective amount of an
agent is not required to cure a disease or condition but will
provide a treatment for a disease or condition such that the onset
of the disease or condition is delayed, hindered or prevented, or
the disease or condition symptoms are ameliorated. The effective
amount may be divided into one, two, or more doses in a suitable
form to be administered at one, two or more times throughout a
designated time period. The specific effective or sufficient amount
will vary with such factors as particular condition being treated,
the physical condition of the patient (e.g., the patient's body
mass, age, or gender), the type of subject being treated, the
duration of the treatment, the nature of concurrent therapy (if
any), and the specific formulations employed and the structure of
the compounds or its derivatives. Effective amount may be
expressed, for example, as the total mass of active component
(e.g., in grams, milligrams or micrograms) or a ratio of mass of
active component to body mass, e.g., as milligrams per kilogram
(mg/kg).
[0070] The terms "application" and "administration" are used
interchangeably herein to mean the application of a molecular
construct or a pharmaceutical composition of the present invention
to a subject in need of a treatment thereof.
[0071] The terms "subject" and "patient" are used interchangeably
herein and are intended to mean an animal including the human
species that is treatable by the molecular construct,
pharmaceutical composition, and/or method of the present invention.
The term "subject" or "patient" intended to refer to both the male
and female gender unless one gender is specifically indicated.
Accordingly, the term "subject" or "patient" comprises any mammal,
which may benefit from the treatment method of the present
disclosure. Examples of a "subject" or "patient" include, but are
not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat,
cow, horse, dog, cat, bird and fowl. In an exemplary embodiment,
the patient is a human. The term "mammal" refers to all members of
the class Mammalia, including humans, primates, domestic and farm
animals, such as rabbit, pig, sheep, and cattle; as well as zoo,
sports or pet animals; and rodents, such as mouse and rat. The term
"non-human mammal" refers to all members of the class Mammalis
except human.
[0072] The present disclosure is based, at least on the
construction of the T-E pharmaceuticals that can be delivered to
target cells, target tissues or organs at increased proportions
relative to the blood circulation, lymphoid system, and other
cells, tissues or organs. When this is achieved, the therapeutic
effect of the pharmaceuticals is increased, while the scope and
severity of the side effects and toxicity is decreased. It is also
possible that a therapeutic effector is administered at a lower
dosage in the form of a T-E molecule, than in a form without a
targeting component. Therefore, the therapeutic effector can be
administered at lower dosages without losing potency, while
lowering side effects and toxicity.
[0073] Diseases that can Benefit from Better Drug Targeting
[0074] Drugs used for many diseases can be improved for better
efficacy and safety, if they can be targeted to the disease sites,
i.e., if they can be localized or partitioned to the disease sites
more favorably than the normal tissues or organs. Following are
primary examples of diseases, in which drugs can be improved if
they can be preferentially distributed to the disease sites or
cells.
[0075] I Immune Disorder
[0076] According to the design of molecular constructs of the
present disclosure, the diseases, conditions, and/or disorders
treatable with the present method is an immune disorder; for
example, an autoimmune disorder that includes, but is not limited
to, psoriasis, systemic lupus erythematosus (SLE), cutaneous lupus,
Sjogren's syndrome, rheumatoid arthritis, psoriatic arthritis,
ankylosing spondylitis, and inflammatory bowel disease.
[0077] Most of the autoimmune diseases, such as rheumatoid
arthritis, systemic lupus erythematosus, Sjogren's syndrome,
psoriasis, Crohn's disease, inflammatory bowel diseases, and others
affect connective tissues. Regardless of the etiological nature,
whether it is environmental, genetic, epigenetic, or their
combinations, the affected tissues are damaged by prolong
inflammatory processes. It is rationalized in this invention that
in bringing anti-inflammatory therapeutic agents, such as
anti-TNF-.alpha., anti-IL-17, anti-BAFF, anti-IL-6,
anti-IL-12/IL-23, to the diseased connective tissues, the
components of the extracellular matrix may be employed as target
antigens. The target antigens that may be considered include the
various types of collagens, laminins, elastins, fibrillins,
fibronectins, and tenascins. Connective tissues fill in nearly all
parts of the human body. However, due to the structural and
functional requirements of the connective tissues in different
locations, the types of those extracellular matrix components are
different, providing excellent choices for target tissue
specificity.
[0078] The advantages of choosing extracellular components over
cell surface antigens for targeting the anti-inflammatory
therapeutic agents are that the choices of selectivity among the
various types of matrix proteins and the abundant amounts of the
extracellular matrix proteins. Furthermore, because cells are not
used as antigenic targets, the potential harmful effects of direct
binding to cells by anti-inflammatory agents can be avoided.
[0079] I-(i) Rheumatoid Arthritis, Psoriatic Arthritis, or
Ankylosing Spondylitis
[0080] Several antibodies against TNF-.alpha., e.g., infliximab and
adalimumab, and fusion proteins of TNF-.alpha. receptor and IgG.Fc
(e.g. etanercept) are approved or in human clinical trials for use
to treat rheumatoid arthritis, ankylosing spondylitis, and other
autoimmune diseases. The extracellular portion the receptor for
interleukin-1 (IL-1), anakinra, is approved for treating rheumatoid
arthritis. Antibodies against the shared p40 protein of IL-12 and
IL-23, e.g., ustekinumab and briakinumab, are approved for
psoriatic arthritis or in trials for rheumatoid arthritis. An
antibody against IL-6 receptor (tocilizumab) is approved for
rheumatoid arthritis and systemic juvenile idiopathic arthritis,
and several antibodies against IL-6, e.g., sarilumab and
olokizumab, are in clinical trials for treating rheumatoid
arthritis. An antibody specific for IL-17 (secukinumab) is approved
for psoriasis and in clinical trials for rheumatoid arthritis and
ankylosing spondylitis.
[0081] While those therapeutic agents can alleviate severe symptoms
better than previously available medications, they cause a range of
serious side effects in some treated patients. For example,
infliximab can cause serious blood disorders, like leukopenia and
thrombocytopenia, serious infections, lymphoma and other solid
tumors, reactivation of hepatitis B and tuberculosis, and other
serious problems. Anakinra causes frequent infections, and severe
side effects on the gastrointestinal and the respiratory tracts and
the blood forming organs. It is important that the serious side
effects of these widely used therapeutic agents be minimized, while
retaining or even enhancing their therapeutic effects.
[0082] In rheumatoid arthritis, joints of the knees, fingers, toes,
and other joints are affected, and in ankylosing spondylitis,
joints of the spine and the sacroiliac joint of the pelvis are
affected. In the diseased joints, the surface of the bones and the
articular cartilage lining the bone surfaces are attacked by the
inflammatory immune components in the joints. The articular
cartilage in the joints is a smooth cartilage that contains an
extracellular matrix. The cartilage is avascular and approximately
60% of the weight is water and the remaining content is composed of
collagens and .alpha.-aggrecan, a proteoglycan, and other matrix
molecules. Collagen II forms the major fibril in the cartilage.
Aggrecan is the second most abundant component in the cartilage.
Collagen XI is bound to the surface of the collagen II fibril
helping to form fibril networks and collagen IX is associated with
collagen II and collagen XI. The cartilage has a large surface and
the .alpha.-aggrecan has a structure and shape like a feather. In
addition to the cartilage formation, the joints have also
ligaments, which connect adjacent bones, such as the cruciate
ligaments, and tendons, which connect muscles to the bones. The
ligaments and tendons are formed by fibrous network of collagen
types I, II, and III, and elastin and fibrillins 1 and 2.
[0083] The present invention rationalizes that the antagonist for
TNF-.alpha., IL-1, and IL-12/IL-23 can be carried to the diseased
joints by using antibody fragments, such as scFv, specific for
collagen II, .alpha.-aggrecan, collagen XI or collagen IX, or
alternatively, collagen I, elastin or fibrillin 1 as the targeting
agent. A preferred anti-collagen II antibody is one that binds to
native collagen II in the joints and does not bind to N-terminal
and C-terminal propeptides, which are cleaved off during fibril
assembly. A preferred anti-aggrecan antibody is one that binds to
whole native .alpha.-aggrecan molecules and does not bind to
fragments that are cleaved off and released into the blood
circulation. By adopting the present molecular construct with scFv
of anti-collagen II as targeting agent, in comparison with regular
IgG against TNF-.alpha., IL-1, and IL12/IL-23, larger proportions
of the present therapeutic agents can be carried to the diseased
sites and less amounts of the therapeutic agents will be present in
other irrelevant, normal tissues, especially, lymphoid organs, and
hence fewer side effects will occur.
[0084] I-(ii) Psoriasis
[0085] Most patients with psoriasis or plaque psoriasis present
inflammatory symptoms primarily in the skin and not in other
tissues and organs. Psoriasis involves mainly keratinocytes in part
of skin in the affected patients. A systematic administration of
monoclonal antibodies anti-TNF-.alpha., anti-IL-12/IL-23, and
anti-IL-17 or anti-IL-17 receptor (anti-IL-17R) or other
anti-inflammatory agents, such as anti-IL6, causes unwanted side
effects, as discussed in the preceding section. The serious adverse
side effects of all these immune modulating antibodies have been
well documented.
[0086] A number of membrane or extracellular proteins, such as
filaggrin, collagen I, which are expressed at much higher levels in
the skin tissues than most of other tissues, probably can be
considered as the target proteins to shuffle therapeutic agents to
the skin. Filaggrin is present in the tight junction between cells
and is probably accessible by antibodies in the diseased tissue
sites. While collagen I is also present in the bone matrix and many
parts of the body, it is present in the dermis layer of the skin in
abundant proportions.
[0087] For damping the inflammatory activity caused by the diseased
keratinocytes, which manifests psoriatic symptoms, it is not
necessary to deliver the anti-inflammatory antibody drugs to be in
contact with the keratinocytes. The keratinocytes are in the
outmost, epidermis layer of the skin; blood vessels, sweat glands,
and collagen fibers are in the middle dermis layer of the skin. The
inner layer is hypodermis, where adipose tissues are. The three
layers of human skin together are 2-3 mm thick. If the
anti-inflammatory antibodies are delivered to the dermis layer by
scFv specific for collagen I, they can diffuse into the other
layers. Or, the antibodies can trap inflammatory cytokines in the
three layers of the skin.
[0088] Several proteins present at the dermo-epidermal junction may
also be employed as targets for carrying therapeutic agents to the
skin. These include type VII collagen, type XVII collagen, and
laminins type 5, 6, or 10. The dermo-epidermal junction is the area
of tissue that joins the epidermal and dermal layers of the skin.
The basal cells in the stratum basale of epidermis connect to the
basement membrane by the anchoring filament of hemidesmosomes. The
cells of the papillary layer of the dermis are attached to the
basement membrane by anchoring fibrils, which consist of type VII
collagen. Type XVII collagen, a transmembrane protein (also
referred to as BP180) expressed on keratinocytes, is a structural
component of hemidesmosomes, multiprotein complexes at the
dermal-epidermal basement membrane zone that mediate adhesion of
keratinocytes to the underlying membrane. Laminins are structural
non-collagenous glycoproteins present in basement membranes. Among
the many types of laminins, types 5, 6, and 10 are specific of the
basal lamina present under stratified epithelia.
[0089] I-(iii) Systemic Lupus Erythematosus (SLE), Cutaneous Lupus,
or Sjogren's Syndrome
[0090] Systemic lupus erythematosus (SLE) is an autoimmune disease
involving multiple autoantigens, such as nucleic acids, histones,
and other nuclear proteins. Sjogren's syndrome is an autoimmune
disease, in which the immune system attacks the exocrine glands,
specifically the salivary and lacrimal glands, which produce saliva
and tears, respectively, resulting the symptoms of dry eyes and dry
mouth, leading to infections and various other problems. Both of
these diseases occur 9 times more frequently in women than in men,
especially in women of child-bearing ages 15 to 35. SLE is a
systemic autoimmune connective tissue disease and affects many
organs and tissues. In general, those tissues and organs, such as
the heart, lungs, bladder, and kidneys, which exhibit elasticity
and can expand and contract, contain collagen network. In several
types of SLE, cutaneous manifestation of inflammatory symptoms is
prominent.
[0091] For more than 50 years, not a single new therapeutic agent
had been developed for SLE, until belimumab, a human monoclonal
antibody specific for BAFF was developed and approved. However, the
therapeutic effect of belimumab for SLE has been considered to be
marginal. Belimumab causes a host of side effects, including more
incidences of serious infections and deaths in the treatment group
than the placebo group. Interestingly, in a phase II trial on
Sjogren's syndrome, belimumab showed more successful results than
in SLE.
[0092] In addition to BAFF, researchers have been searching other
therapeutic targets for SLE. While not a single inflammatory
cytokine has been identified as mainly responsible for the
pathological process in SLE, the expression of a group of genes
known as downstream events of type 1 interferon stimulation, which
is termed "type 1 interferon signature", has been documented in
many studies. The pathogenesis of SLE has been found to be
associated with the activation of toll-like receptors 7 and 9 (TLR
7 and TLR9), which induce the expression of a group of genes
similar to that resulting from the activation by IFN-.alpha..
[0093] Several monoclonal antibodies specific for IFN-.alpha.,
including rontalizumab, sifalimumab, and anifrolumab have been
studied in clinical trials for the treatment of SLE. Since
IFN-.alpha. is involved in many functions, a systemic
administration of an antibody against IFN-.alpha. without localized
targeting to disease sites may render serious side effects.
[0094] I-(iv) Inflammatory Bowel Disease
[0095] Anti-TNF-.alpha. (such as adalimumab) has also been approved
for treating Crohn's disease and ulcerative colitis (a form of
inflammatory bowel disease). However, as described in an earlier
section, the administration of anti-TNF-.alpha. is associated with
a range of series side effects, including severe infectious
diseases and B cell lymphoma. Therefore, in treating patients with
Crohn's disease or ulcerative colitis with anti-TNF-.alpha., it
will be desirable to distribute the administered anti-TNF-.alpha.
in favor of the intestine and colon. It has been found collagen III
and type V are relatively abundant in the connective tissues in the
intestine and bowel.
[0096] II Osteoporosis Disease
[0097] An antibody specific for RANKL (CD254), the ligand of RANK
(RANK, receptor activator of nuclear factor .kappa.B), denosumab,
is approved for the treatment of osteoporosis. The development of
denosumab represents a major advancement in the care for
osteoporosis. However, the administration of denosumab causes
common side effects, such as infections of the urinary and
respiratory tracts, cataracts, constipation, rashes, and joint
pain. It is hence desirable that the therapeutic agent is carried
preferentially to the bone.
[0098] RANKL is a membrane protein, belonging to the tumor necrosis
factor ligand family. RANKL is detected at high levels in the lung,
thymus, and lymph nodes. It is also detected at low levels in the
bone marrow, stomach, peripheral blood, spleen, placenta,
leukocytes, heart, thyroid and skeletal muscle. Since IgG
anti-RANKL, such as denosumab, can serve a therapeutic agent for
osteoporosis, the molecular constructs of this invention should
provide as better therapeutic agents than IgG anti-RANKL.
[0099] Another target for antibodies for the treatment of
osteoporosis is sclerostin, encoded by SOST gene. The glycoprotein
is produced and secreted by osteocytes and negatively regulates
osteoblastic bone formation. The loss or defective mutation of SOST
gene causes progressive bone thickening. A defective mutation in
the SOST gene increases bone formation. Antibodies against
sclerostin cause increased bone formation, bone mineral density,
and stronger bones. The phase I and II clinical trials of two
humanized monoclonal antibodies against sclerostin, blosozumab and
romosozumab, indicated that the antibody treatment is associated
with increased bone mineral density and bone formation and
decreased bone resorption.
[0100] In light of the foregoing discussion, molecular platforms
for constructing the T-E molecules of this invention are provided
in the present disclosure. Detailed discussions relating to the
structure of the molecular construct having the "Fc" configuration
are provided below, as well as the practical applications of each
molecular construct.
[0101] PART I Anti-Inflammatory Molecules with Tissue-Targeting
Functions
[0102] In the broad sense of the Fc-based configuration,
immunoglobulin antibody can serve as the base of a targeting or
effector element, and its corresponding effector or targeting
element can be incorporated at the C-terminal of its two heavy
.gamma. chains in the form of scFv domains. For a typical
"Fc-based" configuration, two-chain IgG.Fc is used as the base of
the molecular platform. Each of the polypeptide chain is fused with
one or two targeting and one or two effector elements, for a total
of two to three elements on each chain. The T-E molecule with an
Fc-based configuration will have a total of four to six elements
(e.g., scFv, growth factor, or cytokines). Optionally, the Fc
portion of the molecular constructs also carries Fc-mediated
effector functions, ADCC, and/or complement-mediated activation.
While in certain other applications, such Fc-mediated effector
functions are avoided.
[0103] In designing the Fc-based molecular constructs, targeting
elements are positioned at the N- or C-terminus. If the effector
elements function by binding to a cell surface component, such as
CD3, CD16a, PD-1, PD-L1, or CTLA-4, they should also be positioned
at the terminus. If the effector elements function by binding to
and neutralizing soluble factors, such as VEGF, TNF-.alpha., IL-17,
or BAFF, they can be positioned between a terminal targeting or
effector element and CH2-CH3.
[0104] In some embodiments of the present disclosure, both the
effector element and the targeting element carried by the CH2-CH3
segment (or CH2-CH3 chain) are mostly comprised of amino acid
residues, and for the sake of discussion, these molecular
constructs are referred to anti-inflammatory molecules with
tissue-targeting functions or anti-inflammatory Fc-based molecular
construct. For example, the effector element may be an antibody
fragment or a soluble receptor, while the targeting element is also
an antibody fragment. Some illustrative structures of this Fc-based
molecular construct are discussed in this section.
[0105] Referring to FIG. 1A, which is a schematic diagram
illustrating an Fc-based molecular construct 800A according to
certain embodiments of the present disclosure. As illustrated, the
Fc-based molecular construct 800A comprises two identical CH2-CH3
chains 810, a first pair of effector elements E1 linked to the
N-termini of the CH2-CH3 chains 810, and a first pair of targeting
elements T1 linked to the C-termini of the CH2-CH3 chains 810. In
this illustrative configuration, both the targeting element T1 and
effector element E1 are antibody fragments.
[0106] In some embodiments, the CH2-CH3 chains are adopted from
human immunoglobulins .gamma.1 or .gamma.4. In general, .gamma.1 is
chosen, when Fc-mediated functions, such as antibody-dependent
cellular cytotoxicity (ADCC) and complement-mediated activity
(inflammatory activation or target cell lysis), are desired. In the
case where Fc-mediated functions are avoided, .gamma.4 is chosen
for constructing the present Fc-based molecular constructs.
[0107] The Fc-based molecular construct 800B illustrated in FIG. 1B
is quite similar to the Fc-based molecular construct 800A of FIG.
1A in structure, except that the two effector elements E1 are
respectively linked to the C-termini of the CH2-CH3 chains 810,
while the two targeting effectors are respectively linked to the
C-termini of the CH2-CH3 chains 810.
[0108] According to certain embodiments, both the effector elements
and targeting elements are linked to the N-termini of the CH2-CH3
chains. For example, when both the effector element and the
targeting element are in the form of single-chain variable
fragments (scFvs), the effector element and the targeting element
may be linked in a tandem or diabody configuration, thereby forming
a bispecific scFv that is linked to the N-terminus of the CH2-CH3
chain.
[0109] The Fc-based molecular construct 800C (FIG. 1C) comprises an
Fc portion, and accordingly, each CH2-CH3 chain 810 has a T1-E1
bispecific scFv linked to the N-terminus thereof.
[0110] As discussed above, the anti-inflammatory Fc-based molecular
constructs can also use a soluble receptor (e.g., the soluble
receptor of TNF-.alpha. or IL-1) as the effector element, according
to certain embodiments. In these cases, the Fc-based molecular
construct 800D (FIG. 1D) may have two effector elements E1
respectively linked to the N-termini of the CH2-CH3 chains 810, and
two targeting elements T1 respectively linked to the C-termini of
the CH2-CH3 chains 810. It is also possible that the effector
elements and the targeting elements are respectively arranged at
the C- and N-termini of the CH2-CH3 chains; see, for example, the
Fc-based molecular construct 800E of FIG. 1E.
[0111] In some examples, the first pair of effector elements or the
first pair of the targeting elements takes a Fab configuration
(i.e., consisting of the V.sub.H-CH1 domain and the
V.sub.L-C.kappa. domain); this Fab fragment is linked to the
N-termini of the CH2-CH3 chains, so that the Fc-based molecular
construct adopts an IgG configuration. In these cases, the pair of
elements that is not in the Fab configuration may be linked to the
C-termini of the pair of CH2-CH3 segments.
[0112] For example, in the Fc-based molecular construct 800F of
FIG. 1F, each of the two targeting elements T1 comprises the
V.sub.H-CH1 domain 820 and the V.sub.L-C.kappa. domain 825, thereby
forming a Fab configuration 830 that is linked to the N-termini of
the CH2-CH3 chains 810, so that the Fc-based molecular construct
800F adopts the IgG configuration. In this case, the pair of
effector elements E1 is linked to the C-termini of the pair of
CH2-CH3 chains 810.
[0113] As described above, the present Fc-based molecular construct
may carry a total of six elements at most. The additional elements
may be a second pair of effector elements or a second pair of
targeting elements.
[0114] According to other embodiment, the Fc-based molecular
construct 900A (FIG. 2A) comprises a second pair of targeting
elements T2. In these cases, the targeting elements T1 and T2 are
linked in a tandem or diabody configuration to form a bispecific
scFv that is linked to the N-terminus of the CH2-CH3 chain 910, and
the effector element E1 is linked to the C-terminus of the CH2-CH3
chain 910.
[0115] According to embodiments exemplified in FIG. 2B, the
Fc-based molecular construct 900B comprises a second pair of
effector elements E2. In these cases, the effector element E1 and
E2 are linked in a tandem or diabody configuration to form a
bispecific scFv that is linked to the N-terminus of the CH2-CH3
chain 910, and the targeting element T1 is linked to the C-terminus
of the CH2-CH3 chain 910.
[0116] Now that the basic structural arrangements of the
anti-inflammatory Fc-based molecular constructs have been discussed
above, certain combinations of particular effector element(s) and
targeting element(s) are provided below for the illustration
purpose.
[0117] According to some embodiments, the effector element is an
scFv specific for TNF-.alpha., and the targeting element is an scFv
specific for collagen II or collagen IX, or .alpha.-aggrecan.
According to some embodiments, each of the two effector elements is
an scFv specific for IL-17, while the targeting element is an scFv
specific for collagen I or collagen VII. Still alternatively, each
of the two effector elements is an scFv specific for BAFF, and the
targeting element is an scFv specific for collagen I or collagen
VII. In some embodiments, each of the two effector elements is an
scFv specific for TNF-.alpha., and the targeting element is an scFv
specific for collagen III or collagen V. In some other embodiments,
the two effector elements are in the form of a Fab specific for
RANKL, and the targeting element is an scFv specific for collagen I
or osteonectin. For example, such molecular construct may take the
configuration of any of those depicted in FIGS. 1A-1C and 1F.
[0118] In some embodiments, the first pair of effector elements
includes an scFv specific for TNF-.alpha. and an scFv specific for
IL-17, while the first pair of targeting elements includes an scFv
specific for collagen II and an scFv specific for collagen IX. In
some alternative embodiments, the first pair of effector elements
includes an scFv specific for TNF-.alpha. and an scFv specific for
IL-17, while the first pair of targeting elements includes an scFv
specific for collagen I and an scFv specific for collagen VII.
Alternatively, each of the two effector elements is an scFv
specific for BAFF, and the targeting element is an scFv specific
for collagen I or collagen VII. Still alternatively, each of the
two effector elements is an scFv specific for RANKL, and the
targeting element is an scFv specific for collagen I or
osteonectin. These molecular constructs may take the configuration
of any of those depicted in FIGS. 1B, 1D, and 1F.
[0119] In certain embodiments, the two effector elements are in the
form of a Fab antibody specific for TNF-.alpha., while the
targeting element is an scFv specific for collagen II or collagen
IX. In some embodiments, the two effector elements are in the form
of a Fab antibody specific for IL-17, and the targeting element is
an scFv specific for collagen I or collagen VII. Alternatively, the
two effector elements are in the form of a Fab antibody specific
for BAFF, and the targeting element is an scFv specific for
collagen I or collagen VII. Still alternatively, the two effector
elements are in the form of a Fab antibody specific for
TNF-.alpha., and the targeting element is an scFv specific for
collagen III or collagen V.
[0120] The essence of this invention is the rationalization and
conception of the specific combination or pairing of the targeting
and effector elements. The adoption of Fc-fusion configuration in
the molecular constructs is a preferred embodiment. It is
conceivable for those skilled in the arts to link the pairs of
targeting and effector elements of this invention employing other
molecular platforms, such as peptides, proteins (e.g., albumin),
polysaccharides, polyethylene glycol, and other types of polymers,
which serve as a structural base for attaching multiple molecular
elements.
[0121] PART II Uses of Anti-Inflammatory Molecules with
Tissue-Targeting Functions
[0122] Another aspect of the present disclosure is directed to the
use of the anti-inflammatory Fc-based molecular constructs
discussed above in PART I.
[0123] As could be appreciated, the description in Part IV-(i)
regarding the rationales underlying the selection of suitable
targeting and effector elements is also applicable in this section.
For example, anti-inflammatory Fc-based molecular constructs used
for treating various immune disorders contain an antibody fragment
(e.g., scFv, Fab, and the like) specific for collagen II, collagen
XI, or .alpha.-aggrecan used as targeting elements and an antibody
fragment (e.g., scFv, Fab, and the like) specific for TNF-.alpha.
and IL-17 as effector elements.
[0124] According to various embodiments of the present disclosure,
the present treatment method involves the administration of a
suitable anti-inflammatory Fc-based molecular construct to a
subject in need of such treatment. Specific examples of
anti-inflammatory Fc-based molecular constructs for treating
various immune disorders, in particular, autoimmune diseases, are
discussed below.
[0125] According to certain embodiments, the present method is used
to treat rheumatoid arthritis, psoriatic arthritis, or ankylosing
spondylitis. In these cases, each effector element of the
anti-inflammatory Fc-based molecular construct is an antibody
fragment specific for TNF-.alpha., IL-12/IL-23, IL-1, IL-17, or
IL-6, while each targeting element is an antibody fragment specific
for collagen II, collagen IX, collagen XI, or .alpha.-aggrecan. For
example, each effector element of the first pair of effector
elements is an scFv specific for TNF-.alpha., while each targeting
element of the first pair of targeting elements is an antibody
fragment specific for collagen II. In other embodiments, the
effector element is an scFv specific for TNF-.alpha., while the
targeting element is an antibody fragment specific for collagen IX.
Alternatively, the effector element is an scFv specific for
TNF-.alpha., while the targeting element is an antibody fragment
specific for .alpha.-aggrecan. According to various embodiments,
the above-mentioned anti-inflammatory Fc-based molecular constructs
may have the configuration of 800A, 800B, or 800C discussed
above.
[0126] Another anti-inflammatory Fc-based molecular construct for
treating rheumatoid arthritis, psoriatic arthritis, or ankylosing
spondylitis comprises two effector elements that are in the form of
a Fab antibody specific for TNF-.alpha.. In these cases, both
targeting elements of the first pair of targeting elements is scFvs
specific for collagen II or scFvs specific for collagen IX.
Configurations of these Fc-based molecular constructs are
illustrated in FIG. 1F, for example.
[0127] The present methods are also applicable in the treatment of
psoriasis. For example, the anti-inflammatory Fc-based molecular
construct may comprise effector elements of an antibody fragment
specific for TNF-.alpha., IL-12/IL-23, or IL-17, and targeting
elements of an antibody fragment specific for collagen I or
collagen VII. According to some embodiments, the effector elements
are scFvs specific for IL-17, while the targeting elements are
scFvs specific for collagen I. Alternatively, the effector elements
are scFvs specific for IL-17, while the targeting elements are
scFvs specific for collagen VII. These anti-inflammatory Fc-based
molecular constructs have the configuration of 800A, 800B, or 800C
discussed above.
[0128] Another anti-inflammatory Fc-based molecular construct for
treating psoriasis comprises two effector elements that are in the
form of a Fab antibody specific for IL-17. In these cases, both
targeting elements of the first pair of targeting elements is scFvs
specific for collagen I or scFvs specific for collagen VII.
Configurations of these Fc-based molecular constructs are
illustrated in FIG. 1F, for example.
[0129] Another set of diseases treatable by the present method
using the anti-inflammatory Fc-based molecular constructs are
systemic lupus erythematosus, cutaneous lupus, or Sjogren's
Syndrome. In these embodiments, each effector is an antibody
fragment specific for BAFF, and each targeting element is an
antibody fragment specific for collagen I or collagen VII. These
anti-inflammatory Fc-based molecular constructs may have the
configuration illustrated in FIGS. 1A to 1C. As could be
appreciated, the pair of effector elements may also take the form
of a Fab antibody specific for BAFF, and the pair of targeting
elements may be scFvs specific for collagen I or collagen VII,
which takes the configuration of the molecular construct 800F
illustrated in FIG. 1F.
[0130] In other embodiments, the present method is used to treat
inflammatory bowel disease, such as Crohn's disease or ulcerative
colitis. In these cases, each effector is an antibody fragment
specific for TNF-.alpha., and each targeting element is an antibody
fragment specific for collagen III or collagen V. Configurations of
these Fc-based molecular constructs are illustrated in FIGS. 1A to
1C, for example. Of course, the pair of effector elements may also
take the form of a Fab antibody specific for TNF-.alpha., while the
pair of targeting elements may be scFvs specific for collagen III
or collagen V, thereby giving the configuration illustrated in FIG.
1F.
[0131] The present anti-inflammatory Fc-based molecular constructs
are also applicable in the treatment of osteoporosis. For example,
the effector elements are antibody fragments specific for RANKL,
while the targeting elements are antibody fragments specific for
collagen I or osteonectin. As could be appreciated, the antibody
fragments specific for RANKL may be scFvs so that the Fc-based
molecular construct has the configuration illustrated in FIG. 1A to
1C, or they may take the form of a Fab so that the Fc-based
molecular construct has the configuration illustrated in FIG.
1F.
[0132] It should be noted that above-examples are given for the
purpose of illustration, and treatments using anti-inflammatory
Fc-based molecular constructs with other T-E combinations are
within the scope of the present disclosure.
EXPERIMENTAL EXAMPLES
Example 1: Construction of Gene Segments Encoding 2-Chain IgG4.Fc
Fusion Protein Containing scFv Specific for Human Collagen II and
scFv Specific for TNF-.alpha.
[0133] Mouse B cell hybridoma II-116B3 producing anti-collagen II
antibody was purchased from Developmental Studies Hybridoma Bank at
the University of Iowa. Poly(A)+ RNA was reverse-transcribed with a
SuperScript III RT-PCR system (Invitrogen, Waltham, USA), and first
strand cDNA was synthesized. The V.sub.H and V.sub.L nucleotide and
amino acid sequences of II-116B3 had not been published. To
determine the sequences of variable regions of II-116B3, cDNA of
V.sub.H and V.sub.L were amplified by PCR using a set of DNA
primers provided by Ig-primer Sets (Novagen, Madison, USA) per the
manufacturer's instructions. The amino acid sequence of V.sub.H and
V.sub.L of II6B3 monoclonal antibody specific for collagen type II
(CII, or COL2) are described in SEQ ID NOs: 3 and 4. The sequences
of V.sub.L and V.sub.H of scFv specific for TNF-.alpha. were those
of V.sub.L and V.sub.H of adalizumab.
[0134] Illustrated below is the configuration of 2-chain IgG4.Fc
fusion protein molecular construct. The scFv1-scFv2-CH2-CH3 (human
.gamma.4) recombinant chain was configured by fusing two scFvs, one
specific for human collagen II and the other specific for human
TNF-.alpha., to the N-terminal of CH2 domain of IgG4.Fc through a
flexible hinge region. The first scFv (specific for collagen II)
had an orientation of V.sub.L-linker-V.sub.H and the second scFv
(specific for TNF-.alpha.) was in V.sub.H-linker-V.sub.L. The
V.sub.L and V.sub.H in each of the two scFv were connected by a
hydrophilic linker, GSTSGSGKPGSGEGSTKG. The two scFvs were
connected via a flexible linker, (GGGGS).sub.3. The sequence of the
recombinant chain in the IgG4.Fc fusion protein molecular construct
illustrated below is described in SEQ ID NO: 5.
[0135] Illustrated below is the configuration of the present
2-chain (scFv .alpha. collagen II)-(scFv .alpha.
TNF-.alpha.)-hIgG4.Fc molecular construct.
Example 2: Expression and Purification of Recombinant 2-Chain (scFv
.alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc Fusion Protein
[0136] In this Example, the gene-encoding sequence was placed in
pcDNA3 expression cassette. Expi293F cells were seeded at a density
of 2.0.times.10.sup.6 viable cells/ml in Expi293F expression medium
and maintained for 18 to 24 hours prior to transfection to ensure
that the cells were actively dividing at the time of transfection.
On the day of transfection, 7.5.times.10.sup.8 cells in 255-ml
medium in a 2-liter Erlenmeyer shaker flask were transfected by
ExpiFectamine.TM. 293 transfection reagent. The transfected cells
were incubated at 37.degree. C. for 16 to 18 hours
post-transfection in an orbital shaker (125 rpm) and the cells were
added ExpiFectamine.TM. 293 transfection enhancer 1 and enhancer 2
to the shaker flask, and incubated for another 7 days. Culture
supernatants were harvested and recombinant 2-chain (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion proteins in the
media were purified using Protein A chromatography. Following
buffer exchange to PBS, the concentration of (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc proteins was determined and
analyzed by SDS-PAGE; see, FIG. 3; II-116B3 (lane 1) and (scFv
.alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc (lane 2) were
analyzed in 10% SDS-PAGE. The Fc-fusion molecular construct was
revealed as the major band at about 80 kDa, consistent with the
expected size.
Example 3: ELISA Analysis of the Binding of Recombinant 2-Chain
(scFv .alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc Fusion
Protein
[0137] To examine the binding ability of recombinant 2-chain (scFv
.alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein to
type II collagen, ELISA assay was performed, using adalimumab and
mouse parental monoclonal antibody II-116B3 for comparison. ELISA
plates were coated with 5 .mu.g/mL of human type II collagen (human
COL2), mouse type II collagen (mouse COL2), and chicken type II
collagen (chick COL2). 1D11 was a human IgG1 antibody against mite
allergen as an isotype control. Recombinant 2-chain (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein, purified
anti-collagen II antibody (II-116B3) and adalimumab were detected
by HRP-conjugated goat anti-human IgG4.Fc, goat anti-mouse IgG.Fc,
and goat anti-human IgG1.Fc, respectively. The ELISA results were
summarized in FIG. 4A.
[0138] To examine the binding ability of 2-chain (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein to human
TNF-.alpha., ELISA assay was performed, along with adalimumab and
mouse parental monoclonal antibody II-116B3. ELISA plates were
coated with 1 .mu.g/mL of human TNF-.alpha. and 1 .mu.g/mL of human
serum albumin as a control. Recombinant 2-chain (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein, purified
anti-collagen II antibody (II-116B3) and Adalimumab were detected
by HRP-conjugated goat anti-human IgG4.Fc, goat anti-mouse IgG.Fc,
and goat anti-human IgG1.Fc, respectively. The results, as
summarized in FIG. 4B, showed that recombinant 2-chain (scFv
.alpha. CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc fusion protein
displayed significant binding activity toward human TNF-.alpha.;
HSA (human serum albumin) was used as control.
Example 4: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing
scFv Specific for Human Collagen II, scFv Specific for TNF-.alpha.
and scFv Specific for Human IL-17
[0139] The scFv1-scFv2-CH2-CH3-scFv3 (human .gamma.4) recombinant
chain was configured by fusing three scFvs, in which the first one
specific for human collagen II and the second one specific for
TNF-.alpha. were fused to the N-terminal of CH2 domain of IgG4.Fc
through a flexible hinge region, while the third one specific for
IL-17 was fused to the C-terminal of CH3 domain.
[0140] The V.sub.H and V.sub.L of the scFv specific for collagen II
were from monoclonal antibody II-116B3; the V.sub.H and V.sub.L of
the scFv specific for TNF-.alpha. were from monoclonal antibody
adalimumab; V.sub.H and V.sub.L of the scFv specific for IL-17 were
from secukinumab. The first scFv (specific for collagen II) had an
orientation of V.sub.L-linker-V.sub.H, the second scFv (specific
for TNF-.alpha.) was in the orientation of V.sub.H-linker-V.sub.L,
and the third scFv (specific for IL-17) was in the orientation of
V.sub.L-linker-V.sub.H. The V.sub.L and V.sub.H in each of the
three scFv were connected by a hydrophilic linker,
GSTSGSGKPGSGEGSTKG. The three scFv were fused via a flexible
linker, (GGGGS).sub.3.
[0141] The sequence of the recombinant chain in the IgG4.Fc fusion
protein molecular construct of the construct illustrated below is
shown in SEQ ID NO: 6. The expression of the constructed genes in
Expi293F cells and the purification of the expressed fusion protein
were performed as in preceding Examples. Characterization of the
new construct was performed with SDS-PAGE and ELISA. The antibody
Secukinumab (Cosentyx) was purchased from Chang Gung Hospital
(Taipei, Taiwan); human IL-17 was from Peprotech (NJ, USA). FIG. 5A
shows the SDA-PAGE results, indicating that the recombinant chain
of the new construct had a size of about 110 kDa, consistent with
the expected size. FIG. 5B shows the ELISA results, indicating that
the present recombinant Fc-fusion protein had the binding activity
to human collagen II, human TNF-.alpha., and human IL-17.
[0142] Illustrated below is the configuration of the present
2-chain (scFv .alpha. CII)-(scFv .alpha.
TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17) molecular construct.
Example 5: Preparation of 2-Chain IgG1.Fc Fusion Protein Containing
TNF-.alpha. Soluble Receptor and scFv Specific for Collagen II
[0143] The (TNF-.alpha. receptor)-CH2-CH3-scFv .alpha. collagen II
(human .gamma.1) recombinant chain was configured by fusing human
TNF-.alpha. receptor, IgG1.Fc, and scFv specific for collagen II.
The sequences of TNF-.alpha. receptor and IgG1.Fc were those of
etanercept. Etanercept and scFv were fused via a flexible linker,
(GGGGS).sub.3. The sequence of the recombinant chain in the IgG4.Fc
fusion protein molecular construct is shown in SEQ ID NO: 7.
[0144] The expression of the constructed genes in Expi293F cells
and the purification of the expressed fusion protein were performed
as in the preceding Examples. Characterization of the new construct
was performed with SDS-PAGE and ELISA. Etanercept (Enbrel) was
purchased from Chang Gung Hospital (Taipei, Taiwan). FIG. 6A shows
the SDS-PAGE results, indicating that the recombinant chain in the
new molecular construct had a size of about 100 kDa, consistent
with the expected size (an Etanercept molecule had a molecular
weight of 150 kDa and one chain of it was about 75 kDa in size; an
scFv was about 27 kDa in size). FIG. 6B shows the ELISA results,
indicating the present molecular construct bound to human
TNF-.alpha., human collagen II, and mouse collagen II.
[0145] Illustrated below is the configuration of the present
2-chain (soluble TNF-.alpha. receptor)-IgG1.CH2-CH3-scFv .alpha.
collagen II molecular construct.
Example 6: Preparation of 2-Chain Fusion Protein Containing Intact
Antibody for Human TNF-.alpha. and scFv Specific for Collagen
II
[0146] The IgG-scFv (human .gamma.1) recombinant chain was
configured by fusing the intact antibody specific for human
TNF-.alpha. and scFv specific for collagen II (illustrated below).
The sequences of intact antibody were those of adalimumab.
Adalimumab and scFv were fused via a flexible linker,
(GGGGS).sub.3.
[0147] The construction of the heavy and light chains of
recombinant genes was built by inserting the two genes into a pG1K
expression cassette with the multiple cloning site. To prepare the
2-chain fusion protein containing intact antibody for human
TNF-.alpha. and scFv specific for collagen type II, transfection of
the expression vectors into Expi293F cells was performed as in
preceding Examples. The amino acid sequence of the heavy chain of
2-chain fusion protein containing intact antibody for human
TNF-.alpha. and scFv specific for collagen type II is indicated in
SEQ ID NO: 8, and the amino acid sequence of the light chain of the
2-chain fusion protein is indicated in SEQ ID NO: 9.
[0148] FIG. 7 shows the SDS-PAGE result, indicating that the heavy
recombinant chain in the present extended IgG molecular construct
had a size of about 75 kDa, consistent with the expected size.
[0149] Illustrated below is the configuration of the present
anti-human TNF-.alpha. in extended IgG configuration with scFv
specific for human collagen II at the C-terminal.
Example 7: Preparation of 2-Chain Fusion Protein Containing Intact
Antibody for Human IL-17 and scFv Specific for Collagen VII
[0150] The IgG-scFv (human .gamma.1) recombinant chain was
configured by fusing the intact antibody specific for human IL-17
and scFv specific for collagen type VII (SEQ ID NO: 20). The
sequences of the intact antibody were those of secukinumab.
Secukinumab and the scFv were fused via a flexible linker,
(GGGGS).sub.3.
[0151] The construction of the heavy and light chains of
recombinant genes was built by inserting the two genes into a pG1K
expression cassette with the multiple cloning site. To prepare the
2-chain fusion protein containing intact antibody for human IL-17
and scFv specific for collagen type VII, transfection of the
expression vectors into Expi293F cells was performed as in the
preceding Example. The amino acid sequence of the heavy chain of
2-chain fusion protein containing intact antibody for human IL-17
and scFv specific for collagen type VII is indicated in SEQ ID NO:
10, and the amino acid sequence of the light chain of the 2-chain
fusion protein is indicated in SEQ ID NO: 11.
[0152] FIG. 8A shows the SDS-PAGE results indicating that the
recombinant heavy chain of the new molecular construct had a size
of 75 kDa, consistent with the expected size. FIG. 8B shows the
ELISA results, indicating that the present molecular construct
bound to human IL17 and human collagen VII. The relatively low
binding to collagen VII was due to a low concentration the antigen
used for coating the ELISA plates
[0153] Illustrated below is the present anti-human IL-17 in
extended IgG configuration with scFv specific for human collagen
VII at the C-terminal.
Example 8: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing
scFv Specific for Human Collagen VII and scFv Specific for BAFF
[0154] cDNA of Mouse B cell hybridoma LH7.2 mAb producing
anti-collagen VII antibody was a gift from Dr. Purdie, cancer
research UK skin tumor laboratory at Queen Mary University of
London. The V.sub.H and V.sub.L sequences of LH7.2 monoclonal
antibody (SEQ ID NOs: 1 and 2) were determined in an earlier
Example. The sequences of V.sub.L and V.sub.H of scFv specific for
BAFF were those of V.sub.L and V.sub.H of belimumab.
[0155] The scFv1-CH2-CH3-scFv2 (human .gamma.4) recombinant chain
(SEQ ID NO: 12) was configured by fusing two scFv, spaced with
IgG4.Fc, one specific for collagen VII and the other specific for
BAFF, to the C-terminal of CH3 domain of IgG4.Fc through a flexible
linker, (GGGGS).sub.3 (illustrated below). The first scFv (specific
for collagen VII) had an orientation of V.sub.L-linker-V.sub.H and
the second scFv (specific for BAFF) was in V.sub.H-linker-V.sub.L.
The V.sub.L and V.sub.H in each of the two scFv were connected by a
hydrophilic linker, GSTSGSGKPGSGEGSTKG.
[0156] The expression of the constructed genes in Expi293F cells
and the purification of the expressed fusion protein were performed
as in an earlier Example. Characterization of the new construct was
performed with SDS-PAGE and ELISA. The antibody Belimumab
(Belynsta) was purchased from Chang Gung Hospital (Taipei); human
BAFF was from GenScript (NJ, USA). FIG. 9A shows the SDA-PAGE
results, indicating that the recombinant chain in the new molecular
construct had a size of about 80-90 kDa, consistent with or
somewhat larger than the expected size. FIG. 9B shows the ELISA
results, indicating that the new construct bound specifically to
human BAFF and human collagen VII.
[0157] Illustrated below is the configuration of the present
2-chain Fc-fusion molecular construct with scFv .alpha. collagen VI
I-IgG4.CH2-CH3-scFv .alpha. BAFF.
Example 9: Preparation of 2-Chain Fusion Protein Containing Intact
Antibody for Human BAFF and scFv Specific for Collagen VII
[0158] The IgG-scFv (human .gamma.1) recombinant chain was
configured by fusing the intact antibody specific for human BAFF
and scFv specific for collagen type VII. The sequences of intact
antibody were those of belimumab. Belimumab and the scFv were fused
via a flexible linker, (GGGGS).sub.3.
[0159] The construction of the heavy and light chains of
recombinant genes was built by inserting the two genes into a pG1K
expression cassette with the multiple cloning site. To prepare the
2-chain fusion protein containing intact antibody for human BAFF
and scFv specific for collagen type VII, transfection of the
expression vectors into Expi293F cells was performed as in
preceding Examples.
[0160] The amino acid sequence of the heavy chain of 2-chain fusion
protein containing intact antibody for human BAFF and scFv specific
for collagen type VII is indicated in SEQ ID NO: 13, and the amino
acid sequence of the light chain of the 2-chain fusion protein is
indicated in SEQ ID NO: 14. FIG. 10A shows the SDS-PAGE results,
indicating that the recombinant heavy chain of the new molecular
construct had a size of about 80 kDa, consistent with the expected
size. FIG. 10B shows the ELISA results, indicating that the new
extended IgG construct bound specifically to human BAFF and human
collagen VII.
[0161] Illustrated below is the configuration of the present
anti-human BAFF extended IgG configuration with scFv specific for
human collagen VII at the C-terminal.
Example 10: Preparation of 2-Chain IgG4.Fc Fusion Protein
Containing scFv Specific for Human Osteonectin and scFv Specific
for RANKL
[0162] Mouse B cell hybridoma AON-1 producing anti-osteonectin
(SPARC) antibody was purchased from Developmental Studies Hybridoma
Bank at the University of Iowa. Poly(A)+ RNA was
reverse-transcribed with a SuperScript III RT-PCR system
(Invitrogen), and the first strand cDNA was synthesized. The
V.sub.H and V.sub.L nucleotide and amino acid sequences of AON-1
had not been published. To determine the sequences of variable
regions of AON-1, cDNA of V.sub.H and V.sub.L were amplified by PCR
using a set of DNA primers provided by Ig-primer Sets (Novagen)
according to the manufacturer's instructions. The V.sub.H and
V.sub.L sequences of AON-1 monoclonal antibody specific for
osteonectin are shown in SEQ ID NOs: 15 and 16. The sequences of
V.sub.L and V.sub.H of scFv specific for RANKL were those of
V.sub.L and V.sub.H of denosumab.
[0163] The scFv1-scFv2-CH2-CH3 (human .gamma.4) recombinant chain
(SEQ ID NO: 17) was configured by fusing two scFv, one specific for
human osteonectin and the other specific for RANKL, to the
N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region
(illustrated below). The first scFv (specific for osteonectin) had
an orientation of V.sub.L-linker-V.sub.H and the second scFv
(specific for RANKL) was in V.sub.H-linker-V.sub.L. The V.sub.L and
V.sub.H in each of the two scFv were connected by a hydrophilic
linker, GSTSGSGKPGSGEGSTKG. The two scFv were fused via a flexible
linker, (GGGGS).sub.3.
[0164] The expression of the constructed genes in Expi293F cells
and the purification of the expressed fusion protein were performed
as in an earlier Example. The characterization of the binding of
the fusion protein to human osteonectin and RANKL by ELISA was
performed as in a preceding Example. Characterization of the new
construct was performed with SDS-PAGE and ELISA. The antibody
Denosumab (Prolia) was purchased from Chang Gung Hospital; human
RANKL and human osteonectin (SPARC) were from GenScript. FIG. 11A
shows the SDS-PAGE results, indicating that the recombinant chain
in the new molecular construct had a size of about 80 kDa. FIG. 11B
shows the ELISA results, indicating that the new Fc-fusion
construct bound specifically to human SPARC and human RANKL.
[0165] Illustrated below is the configuration of the present
2-chain (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4.Fc
molecular construct.
Example 11: Preparation of 2-Chain Fusion Protein Containing Intact
Antibody for Human RANKL and scFv Specific for Human
Osteonectin
[0166] The IgG-scFv (human .gamma.1) recombinant chain was
configured by fusing the intact antibody specific for human RANKL
and scFv specific for human osteonectin. The sequences of intact
antibody were those of denosumab. Denosumab and the scFv were fused
via a flexible linker, (GGGGS).sub.3.
[0167] The construction of the heavy and light chains of
recombinant genes was built by inserting the two genes into a pG1K
expression cassette with the multiple cloning site. To prepare the
2-chain fusion protein containing the intact antibody for human
RANKL and scFv specific for human osteonectin, transfection of the
expression vectors into Expi293F cells was performed as in
preceding Examples.
[0168] The amino acid sequence of the heavy chain of 2-chain fusion
protein containing intact antibody for human RANKL and the scFv
specific for human osteonectin is indicated in SEQ ID NO: 18 and
the amino acid sequence of the light chain of the 2-chain fusion
protein is indicated in SEQ ID NO: 19. FIG. 12A shows the SAS-PAGE
results, indicating that the recombinant heavy chain in the
extended IgG molecular construct had a size of about 80 kDa. FIG.
12B shows the ELISA results, indicating that the new Fc-fusion
construct bound specifically to human SPARC and human RANKL.
[0169] Illustrated below is the configuration of the present
anti-human RANKL in extended IgG configuration with scFv specific
for human osteonectin at the C-terminal.
Example 12: Immunohistologic Chemical Analysis of 2-Chain IgG4.Fc
Fusion Protein Containing scFv Specific for Human Collagen II and
scFv Specific for TNF-.alpha. in Binding to Joint Cartilage
[0170] Immunohistologic analysis was performed in the histology
core facility of Genomics Research Center, Academia Sinica to
examine whether the molecular construct, 2-chain (scFv .alpha.
collagen II)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc (the configuration
illustrated in an earlier Example), had affinity for binding to
cartilage. Mouse bone and cartilage samples were obtained from
FVB/N mice sacrificed by using CO.sub.2. Femur connected with tibia
and knee femoral ends were harvested and fixed with 10% neutral
buffered formaldehyde at room temperature for 48 hours. Samples
were then decalcified in 10% EDTA, pH 7.4, for 7 days, with daily
renewal of the solution. After decalcification, samples were
post-fixed in 10% neutral buffered formaldehyde at room temperature
for 24 hours and stored in 70% ethanol at 4.degree. C. until
dehydration by ASP6025 Tissue Processor (Leica) and paraffin
embedding.
[0171] Safranin 0 staining was performed according to the protocol
described in Schmitz et al., 2010. For immunostaining,
3-.mu.m-thick sections were deparaffinized and rehydrated using
Leica AutoStainer XL, followed by staining procedures described in
the Tyramide Signal Amplification Biotin kit (PerkinElmer). In
brief, sections were quenched for endogenous peroxidase activity in
3% H.sub.2O.sub.2 for 15 minutes, followed by antigen retrieval
with 1 mg/mL hyaluronidase (Sigma Aldrich) in 37.degree. C. for 20
minutes and 20 .mu.g/mL proteinase k (TOOLS) at room temperature
for 10 minutes. Sections were next blocked in TNB buffer of the TSA
kit. For staining with mouse II-116B3 antibody, additional mouse
IgG blocking reagent (Vector Laboratories) was added preceding TNB
blocking. Both primary antibodies II-116B3 and (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc were used at 50 .mu.g/mL.
Goat anti-mouse IgG Fc and goat anti-human IgG Fc (Jackson
ImmunoResearch) were use at 1.6 .mu.g/mL for incorporating HRP,
which reacted with the subsequently added biotin-tyramide. The
biotin labels were then probed with streptavidin-HRP and
chromogenically visualized with diaminobenzidine substrate
(BioGenex). Sections were counterstained using hematoxylin and
mounted with Leica CV5030 Coverslipper.
[0172] FIGS. 13 panel A to 13 panel C showed the immunostaining of
mouse epiphyseal bone with monoclonal antibodies II-116B3 (FIG. 13
panel A), adalimumab (FIG. 13 panel B), and (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4Fc, i.e., the configuration
illustrated in an earlier Example (FIG. 13 panel C), followed by
HRP-labeled goat anti-mouse or goat anti-human secondary antibodies
and tyramide amplification. Type II collagen was revealed at
epiphyseal articular cartilage (AC) and growth plate (GP) in FIG.
13 panel A and FIG. 13 panel C. The results showed that the
anti-collagen II monoclonal antibody II-116B3 stained the AC and GP
parts prominently (FIG. 13 panel A), while adalimumab had no
significant staining (FIG. 13 panel B). The present construct also
stained positively (FIG. 13 panel C). The positive staining
originally revealed in brown color was converted to black/gray. The
scale bar represents 250 .mu.m.
Example 13: Immunohistologic Chemical Analysis of 2-Chain IgG1.Fc
Fusion Protein Containing scFv Specific for Collagen II and scFv
Specific for TNF-.alpha. and scFv Specific for IL-17 in Binding to
Joint Cartilage
[0173] The preparation of tissue thin sections, staining with the
molecular construct, 2-chain (scFv .alpha. CII)-(scFv .alpha.
TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17) (the configuration
illustrated in an earlier Example) and controls were performed as
in the preceding Examples.
[0174] FIGS. 13 panel D to 13 panel E showed the immunostaining of
collagen II in mouse epiphyseal bone. 3-.mu.m thick sections of the
femoral end of mouse knee were stained with monoclonal antibodies,
an anti-IL17a mouse antibody (purchased from PeproTech, NJ, USA)
(FIG. 13 panel D), and the present construct 2-chain (scFv .alpha.
CII)-(scFv .alpha. TNF-.alpha.)-hIgG4.Fc-(scFv .alpha. IL-17) (FIG.
13 panel E), followed by HRP-labeled goat anti-mouse or goat
anti-human secondary antibodies and tyramide amplification. The
results showed that anti-IL17 monoclonal antibody had no
significant staining, while the present construct had moderately
positive staining.
Example 14: Immunohistologic Chemical Analysis of 2-Chain IgG1.Fc
Fusion Protein Containing TNF-.alpha. Soluble Receptor and scFv
Specific for Collagen II in Binding to Joint Cartilage
[0175] The preparation of tissue thin sections, staining with the
molecular construct, 2-chain (soluble TNF-.alpha.
receptor)-IgG1.CH2-CH3-scFv .alpha. collagen II (the configuration
illustrated in an earlier Example) and controls were performed as
in the preceding Example.
[0176] The staining procedure was the same as in preceding
examples. The mouse epiphyseal bone tissue section samples were
from the same batch. The results showed that the positive control
II-116B3 stained strongly (FIG. 14 panel A), etanercept had no
significant staining (FIG. 14 panel B), and the present construct
stained collagen II-containing components AC and GP positively
(FIG. 14 panel C).
Example 15: Bio-Distribution of Recombinant 2-Chain IgG4.Fc Fusion
Protein Containing scFv Specific for Human Osteonectin (SPARC) and
scFv Specific for RANKL Using In Vivo Imaging System
[0177] A Dylight 680 Antibody Labeling Kit (Thermo Scientific) was
used to conjugate denosumab and (scFv .alpha. SPARC)-(scFv .alpha.
RANKL)-hIgG4Fc (scheme 67 of Example 60), according to the
manufacturer's instructions. 8 to 10-week-old BALB/c mice were
injected intravenously with PBS or 40 .mu.g labeled antibodies. At
various time points, mice were anaesthetized with isoflurane in
O.sub.2 and placed in the IVIS Spectrum In Vivo Imaging System
(PerkinElmer) with a supine position. Fluorescent images were
captured with ex/em=675/720, using the Living Image Software
V3.2.
[0178] To investigate the targeting effect of (scFv .alpha.
SPARC)-(scFv .alpha. RANKL)-hIgG4Fc, tissue distribution of
antibodies in mice was compared, through observing fluorescent
signals from the abdominal aspect. Fluorescent images from BALB/c
mice were acquired and analyzed at 30 minutes, 3 hours, and 28
hours after the administration of DyLight 680-conjugated
antibodies. 30 minutes after the injection, the penetration of
anti-SPARC, (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc, and
BoneTag into limbs was greater than that observed with denosumab.
In addition to bladder accumulation, the distribution of denosumab,
anti-SPARC, and (scFv .alpha. SPARC)-(scFv .alpha. RANKL)-hIgG4Fc
was more dispersed after 3 hours, while BoneTag was restricted to
the head and the limbs. Bone structures were clearly resolved with
remaining anti-SPARC 28 hours after the antibody
administration.
[0179] FIG. 15 showed bio-distribution of fluorescence-labeled
antibodies in vivo. BALB/c mice were intravenously injected with
PBS (1), denosumab (2), anti-SPARC mAb (3), (scFv .alpha.
SPARC)-(scFv .alpha. RANKL)-hIgG4Fc (4), and BoneTag (5). Images
were captured at indicated time points using IVIS Spectrum imager
and analyzed with Living Image software. Spectral unmixing was
performed to distinguish tissue autofluorescence from DyLight 680
signals. The results showed that in comparison to denosumab, the
present construct was distributed more similarly to anti-SPARC
monoclonal antibody at 30 minutes after treatment. At longer time
points, the distribution was influenced by half-lives of the
reagents. Anti-SPARC and denosumab were both antibodies and had
similar serum half-lives.
[0180] It will be understood that the above description of
embodiments is given by way of example only and that various
modifications may be made by those with ordinary skill in the art.
The above specification, examples and data provide a complete
description of the structure and use of exemplary embodiments of
the invention. Although various embodiments of the invention have
been described above with a certain degree of particularity, or
with reference to one or more individual embodiments, those with
ordinary skill in the art could make numerous alterations to the
disclosed embodiments without departing from the spirit or scope of
this invention.
Sequence CWU 1
1
201112PRTArtificial Sequence1F10-VH 1Gln Ile Gln Leu Val Gln Ser
Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His
Trp Val Lys Gln Ala Pro Gly Glu Gly Leu Lys Trp Met 35 40 45 Gly
Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55
60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr
Phe Cys 85 90 95 Tyr Tyr Gly Ser Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ala 100 105 110 2111PRTArtificial Sequence1F10-VL 2Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30 Gly Asp Gly Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Phe Ala Ala Ser Asn Leu Lys Ser
Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Tyr Pro Trp Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 3134PRTArtificial
SequenceII6B3-VH 3Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly
Ile Thr Phe Ser Asp Tyr 20 25 30 Trp Met Asp Trp Val Arg Gln Ser
Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Leu Lys
Ser Ser Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95
Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr
Pro 115 120 125 Leu Val Pro Gly Ser Leu 130 4129PRTArtificial
SequenceII6B3-VL 4Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser
Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95
Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser
Lys 115 120 125 Leu 5741PRTArtificial Sequence(scFv anti-collagen
II)-(scFv anti-TNF-alpha)-hIgG4.Fc 5Asp Val Leu Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Phe
Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys
Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro
Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Lys Leu Glu
Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Met Lys
Leu Ser Cys Val Ala Ser Gly Ile Thr Phe 145 150 155 160 Ser Asp Tyr
Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 165 170 175 Glu
Trp Val Ala Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His 180 185
190 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
195 200 205 Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu
Asp Thr 210 215 220 Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe
Val Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala
Ala Lys Thr Thr Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu 260 265 270 Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu 275 280 285 Ser Cys Ala
Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp 290 295 300 Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr 305 310
315 320 Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg
Phe 325 330 335 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu
Gln Met Asn 340 345 350 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Lys Val Ser 355 360 365 Tyr Leu Ser Thr Ala Ser Ser Leu Asp
Tyr Trp Gly Gln Gly Thr Leu 370 375 380 Val Thr Val Ser Ser Gly Ser
Thr Ser Gly Ser Gly Lys Pro Gly Ser 385 390 395 400 Gly Glu Gly Ser
Thr Lys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 405 410 415 Ser Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 420 425 430
Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 435
440 445 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser
Gly 450 455 460 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu 465 470 475 480 Thr Ile Ser Ser Leu Gln Pro Glu Asp Val
Ala Thr Tyr Tyr Cys Gln 485 490 495 Arg Tyr Asn Arg Ala Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu 500 505 510 Ile Lys Arg Ala Ser Pro
Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 515 520 525 Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 530 535 540 Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 545 550 555
560 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
565 570 575 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser 580 585 590 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 595 600 605 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser 610 615 620 Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 625 630 635 640 Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 645 650 655 Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 660 665 670 Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 675 680
685 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
690 695 700 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 705 710 715 720 Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser 725 730 735 Leu Ser Leu Gly Lys 740
61015PRTArtificial Sequencefusion protein containing scFv specific
for human collagen type II, scFv specific for TNF-alpha and scFv
specific for human IL-17 6Asp Val Leu Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Phe Cys Arg
Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 110 Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
Glu Gly Ser 115 120 125 Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly
Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Met Lys Leu Ser Cys
Val Ala Ser Gly Ile Thr Phe 145 150 155 160 Ser Asp Tyr Trp Met Asp
Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 165 170 175 Glu Trp Val Ala
Glu Ile Arg Leu Lys Ser Ser Asn Tyr Ala Thr His 180 185 190 Tyr Ala
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 195 200 205
Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr 210
215 220 Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp
Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr
Thr Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu 260 265 270 Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Arg Ser Leu Arg Leu 275 280 285 Ser Cys Ala Ala Ser Gly
Phe Thr Phe Asp Asp Tyr Ala Met His Trp 290 295 300 Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr 305 310 315 320 Trp
Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe 325 330
335 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn
340 345 350 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys
Val Ser 355 360 365 Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly
Gln Gly Thr Leu 370 375 380 Val Thr Val Ser Ser Gly Ser Thr Ser Gly
Ser Gly Lys Pro Gly Ser 385 390 395 400 Gly Glu Gly Ser Thr Lys Gly
Asp Ile Gln Met Thr Gln Ser Pro Ser 405 410 415 Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 420 425 430 Ser Gln Gly
Ile Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 435 440 445 Lys
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly 450 455
460 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
465 470 475 480 Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr
Tyr Cys Gln 485 490 495 Arg Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Val Glu 500 505 510 Ile Lys Arg Ala Ser Gly Gly Ser Pro
Pro Cys Pro Ser Cys Pro Ala 515 520 525 Pro Glu Phe Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 530 535 540 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 545 550 555 560 Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 565 570 575
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 580
585 590 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln 595 600 605 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly 610 615 620 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 625 630 635 640 Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr 645 650 655 Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 660 665 670 Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 675 680 685 Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 690 695 700
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 705
710 715 720 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 725 730 735 Ser Leu Ser Leu Ser Leu Gly Lys Gly Thr Gly Gly
Gly Gly Ser Gly 740 745 750 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Ile Val Leu Thr Gln Ser 755 760 765 Pro Gly Thr Leu Ser Leu Ser Pro
Gly Glu Arg Ala Thr Leu Ser Cys 770 775 780 Arg Ala Ser Gln Ser Val
Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln 785 790 795 800 Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg 805 810 815 Ala
Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 820 825
830 Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
835 840 845 Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Cys Thr Phe Gly Gln
Gly Thr 850 855 860 Arg Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser
Gly Lys Pro Gly 865 870 875 880 Ser Gly Glu Gly Ser Thr Lys Gly Glu
Val Gln Leu Val Glu Ser Gly 885 890 895 Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala 900 905 910 Ser Gly Phe Thr Phe
Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala 915 920 925 Pro Gly Lys
Gly Leu Glu Trp Val Ala Ala Ile Asn Gln Asp Gly Ser 930 935 940 Glu
Lys Tyr Tyr Val Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 945 950
955 960 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg
Val 965 970 975 Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Asp Tyr Tyr
Asp Ile Leu 980 985 990 Thr Asp Tyr Tyr Ile His Tyr Trp Tyr Phe Asp
Leu Trp Gly Arg Gly 995 1000 1005 Thr Leu Val Thr Val Ser Ser 1010
10157735PRTArtificial Sequencefusion protein
containing TNF-alpha soluble receptor and scFv specific for
collagen type II 7Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro
Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln
Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser Pro Gly Gln His
Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp
Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val
Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln
Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95
Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100
105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val
Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro
Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp
Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile
Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser
Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190 His Leu Pro Gln
Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205 Pro Glu
Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220
Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 225
230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345
350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro
Gly Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 465 470
475 480 Gly Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val
Ser 485 490 495 Leu Gly Asp Gln Ala Ser Ile Phe Cys Arg Ser Ser Gln
Ser Ile Val 500 505 510 His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro Gly 515 520 525 Gln Ser Pro Lys Val Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser Gly 530 535 540 Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu 545 550 555 560 Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe 565 570 575 Gln Gly
Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu 580 585 590
Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu 595
600 605 Gly Ser Thr Lys Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly
Leu 610 615 620 Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala
Ser Gly Ile 625 630 635 640 Thr Phe Ser Asp Tyr Trp Met Asp Trp Val
Arg Gln Ser Pro Glu Lys 645 650 655 Gly Leu Glu Trp Val Ala Glu Ile
Arg Leu Lys Ser Ser Asn Tyr Ala 660 665 670 Thr His Tyr Ala Glu Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 675 680 685 Asp Ser Lys Ser
Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu 690 695 700 Asp Thr
Gly Ile Tyr Tyr Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr 705 710 715
720 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr 725
730 735 8719PRTArtificial Sequenceheavy chain of fusion protein
containing intact antibody for human TNF-alpha and scFv specific
for collagen type II 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp
Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Asp
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Arg Asn Gln Val
Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly
Lys Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460
Gly Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro 465
470 475 480 Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Ile Thr
Phe Ser 485 490 495 Asp Tyr Trp Met Asp Trp Val Arg Gln Ser Pro Glu
Lys Gly Leu Glu 500 505 510 Trp Val Ala Glu Ile Arg Leu Lys Ser Ser
Asn Tyr Ala Thr His Tyr 515 520 525 Ala Glu Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys 530 535 540 Ser Ser Val Tyr Leu Gln
Met Asn Asn Leu Arg Ala Glu Asp Thr Gly 545 550 555 560 Ile Tyr Tyr
Cys Thr Pro Leu Thr Gly Gly Phe Val Tyr Trp Gly Gln 565 570 575 Gly
Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Gly Ser Thr Ser 580 585
590 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Val
595 600 605 Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
Asp Gln 610 615 620 Ala Ser Ile Phe Cys Arg Ser Ser Gln Ser Ile Val
His Ser Asn Gly 625 630 635 640 Asn Thr Tyr Leu Glu Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Lys 645 650 655 Val Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro Asp Arg 660 665 670 Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg 675 680 685 Val Glu Ala
Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His 690 695 700 Val
Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 705 710 715
9214PRTArtificial Sequencelight chain of fusion protein containing
intact antibody for human TNF-alpha and scFv specific for collagen
type II 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala
Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly
Glu Cys 210 10721PRTArtificial Sequenceheavy chain of fusion
protein containing intact antibody for human IL-17 and scFv
specific for collagen type VII 10Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala
Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val Gly Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Val Arg Asp Tyr Tyr Asp Ile Leu Thr Asp Tyr Tyr
Ile His Tyr Trp 100 105 110 Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu
Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185
190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310
315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435
440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly
Gly 450 455 460 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Met Leu Val
Glu Ser Gly 465 470 475 480 Gly Gly Leu Val Lys Pro Gly Gly Ser Leu
Lys Leu Ser Cys Ala Ala 485 490 495 Ser Gly Phe Thr Phe Ser Ser Tyr
Ala Met Ser Trp Val Arg Gln Thr 500 505 510 Pro Glu Lys Arg Leu Glu
Trp Val Ala Thr Ile Thr Ser Gly Gly Asp 515 520 525 Tyr Thr Phe Tyr
Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 530 535 540 Asp Asn
Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Arg Arg Ser 545 550 555
560 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Gly Glu Ile Gly
Tyr
565 570 575 Gly Ser Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr 580 585 590 Val Ser Ala Gly Ser Thr Ser Gly Ser Gly Lys Pro
Gly Ser Gly Glu 595 600 605 Gly Ser Thr Lys Gly Asp Val Gln Ile Thr
Gln Ser Pro Ser Tyr Leu 610 615 620 Ala Ala Ser Pro Gly Glu Thr Ile
Thr Ile Asn Cys Arg Ala Ser Lys 625 630 635 640 Ser Ile Ser Lys Tyr
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr 645 650 655 Asn Lys Leu
Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro 660 665 670 Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 675 680
685 Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His
690 695 700 Asn Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 705 710 715 720 Arg 11215PRTArtificial Sequencelight chain
of fusion protein containing intact antibody for human IL-17 and
scFv specific for collagen type VII 11Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
Ser Pro 85 90 95 Cys Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 12742PRTArtificial
Sequencefusion protein containing scFv specific for human collagen
type VII and scFv specific for BAFF 12Asp Val Gln Ile Thr Gln Ser
Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile
Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30 Leu Ala Trp
Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr
Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr
Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly
Ser Thr Lys Gly Glu Val 115 120 125 Met Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly Ser Leu 130 135 140 Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Ser Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr 165 170 175 Ile
Thr Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly 180 185
190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
195 200 205 Met Ser Ser Arg Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
Ala Arg 210 215 220 His Gly Glu Ile Gly Tyr Gly Ser Ser Ala Trp Phe
Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala
Ala Ser Gly Gly Ser Pro Pro 245 250 255 Cys Pro Ser Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 290 295 300 Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310
315 320 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val 325 330 335 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys 340 345 350 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser 355 360 365 Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 370 375 380 Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 420 425 430
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 435
440 445 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 450 455 460 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Gly Gly 465 470 475 480 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Val Gln 485 490 495 Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser Ser Val Arg 500 505 510 Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Asn Asn Asn Ala Ile Asn 515 520 525 Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile 530 535 540 Ile Pro
Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln Gly Arg 545 550 555
560 Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser Met Glu Leu
565 570 575 Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Ser 580 585 590 Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser
Pro Trp Gly Arg 595 600 605 Gly Thr Met Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 610 615 620 Gly Ser Gly Gly Gly Gly Ser Ala
Phe Ser Ser Glu Leu Thr Gln Asp 625 630 635 640 Pro Ala Val Ser Val
Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln 645 650 655 Gly Asp Ser
Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro 660 665 670 Gly
Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser 675 680
685 Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser
690 695 700 Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys 705 710 715 720 Ser Ser Arg Asp Ser Ser Gly Asn His Trp Val
Phe Gly Gly Gly Thr 725 730 735 Glu Leu Thr Val Leu Gly 740
13717PRTArtificial Sequenceheavy chain of fusion protein containing
intact antibody for human BAFF and scFv specific for collagen type
VII 13Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ser 1 5 10 15 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
Asn Asn Asn 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Met Phe Gly Thr
Ala Lys Tyr Ser Gln Asn Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr
Ala Asp Glu Ser Thr Gly Thr Ala Ser 65 70 75 80 Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser
Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro 100 105 110 Trp
Gly Arg Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 210 215 220 Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245
250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370
375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys Gly
Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser
Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val 465 470 475 480 Lys
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 485 490
495 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg
500 505 510 Leu Glu Trp Val Ala Thr Ile Thr Ser Gly Gly Asp Tyr Thr
Phe Tyr 515 520 525 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys 530 535 540 Asn Thr Leu Tyr Leu Gln Met Ser Ser Arg
Arg Ser Glu Asp Thr Ala 545 550 555 560 Met Tyr Tyr Cys Ala Arg His
Gly Glu Ile Gly Tyr Gly Ser Ser Ala 565 570 575 Trp Phe Ala Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly 580 585 590 Ser Thr Ser
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 595 600 605 Gly
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro 610 615
620 Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys
625 630 635 640 Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn
Lys Leu Leu 645 650 655 Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile
Pro Ser Arg Phe Ser 660 665 670 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Glu 675 680 685 Pro Glu Asp Phe Ala Met Tyr
Tyr Cys Gln Gln His Asn Glu Tyr Pro 690 695 700 Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg 705 710 715 14214PRTArtificial
Sequencelight chain of fusion protein containing intact antibody
for human BAFF and scFv specific for collagen type VII 14Ser Ser
Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15
Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20
25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile
Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Ser Ser
Arg Asp Ser Ser Gly Asn His 85 90 95 Trp Val Phe Gly Gly Gly Thr
Glu Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Asn Pro Thr Val
Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala
Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly 145 150
155 160 Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210
15140PRTArtificial SequenceVH of SPARC 15Gln Val Gln Leu Gln Gln
Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Thr Ile
His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Thr
Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Leu Ala Phe Gly Ile Ile Thr Thr Val
Val Arg Tyr Phe Asp 100 105 110 Val Trp Gly Thr Gly Thr Thr Val Thr
Val Ser Ser Ala Lys Thr Thr 115 120 125 Ala Pro Ser Val Tyr Pro Leu
Ala Pro Gly Ser Leu 130 135 140 16125PRTArtificial SequenceVL of
SPARC 16Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val
Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile
His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser
Pro Gln Leu Leu Val
35 40 45 Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His
Phe Trp Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe
Pro Pro Ser Ser Lys Leu Gly 115 120 125 17751PRTArtificial
SequenceFc fusion protein containing scFv specific for human
osteonectin and scFv specific for RANKL 17Asp Ile Gln Met Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr
Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45
Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser
Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
Arg Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu
Gly Ser Thr Lys Gly Gln Val 115 120 125 Gln Leu Gln Gln Ser Asp Ala
Glu Leu Val Lys Pro Gly Ala Ser Val 130 135 140 Lys Ile Ser Cys Lys
Val Ser Gly Tyr Thr Phe Thr Asp His Thr Ile 145 150 155 160 His Trp
Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Tyr 165 170 175
Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly 180
185 190 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Thr Tyr Met
Gln 195 200 205 Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
Cys Ala Arg 210 215 220 Leu Ala Phe Gly Ile Ile Thr Thr Val Val Arg
Tyr Phe Asp Val Trp 225 230 235 240 Gly Thr Gly Thr Thr Val Thr Val
Ser Ser Ala Lys Thr Thr Gly Gly 245 250 255 Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln 260 265 270 Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 275 280 285 Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 290 295 300
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile 305
310 315 320 Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
Gly Arg 325 330 335 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln Met 340 345 350 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Lys Asp 355 360 365 Pro Gly Thr Thr Val Ile Met Ser
Trp Phe Asp Pro Trp Gly Gln Gly 370 375 380 Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Ser Thr Ser Gly 385 390 395 400 Ser Gly Lys
Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Val 405 410 415 Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 420 425
430 Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg Tyr Leu Ala
435 440 445 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr Gly 450 455 460 Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser Gly Ser Gly 465 470 475 480 Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro Glu Asp 485 490 495 Phe Ala Val Phe Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro Arg Thr Phe 500 505 510 Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Ala Ser Gly Gly Ser Pro 515 520 525 Pro Cys Pro
Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 530 535 540 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 545 550
555 560 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu 565 570 575 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys 580 585 590 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser 595 600 605 Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys 610 615 620 Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 625 630 635 640 Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 645 650 655 Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 660 665 670
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 675
680 685 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 690 695 700 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg 705 710 715 720 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 725 730 735 His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 740 745 750 18721PRTArtificial
Sequenceheavy chain of fusion protein containing intact antibody
for human RANKL and scFv specific for human osteonectin 18Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Gly Thr Thr
Val Ile Met Ser Trp Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150
155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys Gly Gly Gly Ala Ser Gly
Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Gln Val Gln Leu Gln
Gln Ser Asp Ala Glu Leu Val Lys 465 470 475 480 Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe 485 490 495 Thr Asp His
Thr Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu 500 505 510 Glu
Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn 515 520
525 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
530 535 540 Thr Thr Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser
Ala Val 545 550 555 560 Tyr Phe Cys Ala Arg Leu Ala Phe Gly Ile Ile
Thr Thr Val Val Arg 565 570 575 Tyr Phe Asp Val Trp Gly Thr Gly Thr
Thr Val Thr Val Ser Ser Ala 580 585 590 Lys Thr Thr Gly Ser Thr Ser
Gly Ser Gly Lys Pro Gly Ser Gly Glu 595 600 605 Gly Ser Thr Lys Gly
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu 610 615 620 Ser Ala Ser
Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly 625 630 635 640
Asn Ile His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser 645
650 655 Pro Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val
Pro 660 665 670 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser
Leu Lys Ile 675 680 685 Asn Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr
Tyr Cys Gln His Phe 690 695 700 Trp Ser Thr Pro Phe Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys 705 710 715 720 Arg 19215PRTArtificial
Sequencelight chain of fusion protein containing intact antibody
for human RANKL and scFv specific for human osteonectin 19Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Phe Tyr Cys
Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150
155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
20260PRTArtificial SequenceCVII scFv 20Asp Val Gln Ile Thr Gln Ser
Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile
Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr 20 25 30 Leu Ala Trp
Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr
Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr
Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Gly Ser Thr Ser 100 105 110 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly
Ser Thr Lys Gly Glu Val 115 120 125 Met Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly Ser Leu 130 135 140 Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Ser Trp Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr 165 170 175 Ile
Thr Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val Lys Gly 180 185
190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln
195 200 205 Met Ser Ser Arg Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
Ala Arg 210 215 220 His Gly Glu Ile Gly Tyr Gly Ser Ser Ala Trp Phe
Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ala
Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Gly Ser Cys 260
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