U.S. patent application number 15/159863 was filed with the patent office on 2016-11-24 for molecular constructs for treating infectious diseases.
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, Li-Yun DU.
Application Number | 20160340427 15/159863 |
Document ID | / |
Family ID | 57318912 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160340427 |
Kind Code |
A1 |
CHANG; Tse-Wen ; et
al. |
November 24, 2016 |
Molecular constructs for treating infectious diseases
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
City, TW) ; CHU; Hsing-Mao; (Taipei City, TW)
; CHEN; Jou-Han; (Taipei City, TW) ; DU;
Li-Yun; (Keelung City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunwork Inc. |
Taipei City |
|
TW |
|
|
Assignee: |
Immunwork Inc.
Taipei City
TW
|
Family ID: |
57318912 |
Appl. No.: |
15/159863 |
Filed: |
May 20, 2016 |
Related U.S. Patent Documents
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Application
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Patent Number |
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14997764 |
Jan 18, 2016 |
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15159863 |
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14997827 |
Jan 18, 2016 |
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14997764 |
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14997849 |
Jan 18, 2016 |
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14997827 |
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14997874 |
Jan 18, 2016 |
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14997849 |
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62308349 |
Mar 15, 2016 |
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62213012 |
Sep 1, 2015 |
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62164400 |
May 20, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/74 20130101;
A61K 47/64 20170801; C07K 16/2839 20130101; A61K 47/6883 20170801;
C07K 2317/31 20130101; C07K 2317/35 20130101; C07K 2317/64
20130101; A61P 31/04 20180101; C07K 7/08 20130101; C07K 16/283
20130101; A61P 25/00 20180101; C07K 2317/622 20130101; A61P 25/28
20180101; C07K 2319/33 20130101; A61K 47/6849 20170801; C07K
16/2881 20130101; C07K 17/06 20130101; C07K 16/1027 20130101; C07K
2319/70 20130101; C07K 2317/55 20130101; A61K 31/137 20130101; C07K
16/1203 20130101; C07K 14/001 20130101; A61K 47/65 20170801; C07K
16/18 20130101; A61K 47/60 20170801; A61P 31/12 20180101; C07K
2317/76 20130101; C07K 14/565 20130101; C07K 17/02 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/12 20060101 C07K016/12; C07K 14/565 20060101
C07K014/565; C07K 16/10 20060101 C07K016/10 |
Claims
1. A molecular construct comprising, a pair of CH2-CH3 segments of
an IgG.Fc; a pair of effector elements, wherein each effector
element is an antibody fragment specific for CD16b or CD32; and a
pair of targeting elements, wherein each targeting element is an
antibody fragment specific for a viral protein or a bacterial
protein, wherein, when the pair of effector elements is linked to
the N-termini of the pair of CH2-CH3 segments, then the pair of
targeting elements is linked to the C-termini of the pair of
CH2-CH3 segments, and vice versa, or when the pair of effector
elements and the pair of targeting elements are both in the form of
single-chain variable fragments (scFvs), then the pair of targeting
elements is linked to the N-termini of the 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.1 or .gamma.4
immunoglobulin.
3. The molecular construct of claim 1, wherein when the pair of
effector elements is in the form of an antigen-binding fragment
(Fab), and the 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 the
molecular construct adopts an extended IgG configuration.
4. The molecular construct of claim 1, wherein the viral protein is
F protein of respiratory syncytia virus (RSV), gp120 protein of
human deficiency virus type 1 (HIV-1), hemagglutinin A (HA) protein
of influenza A virus, or glycoprotein of cytomegalovirus.
5. The molecular construct of claim 1, wherein the bacterial
protein is endotoxin of Gram(-) bacteria, surface antigen of
Clostridium difficile, lipoteichoic acid of staphylococcus aureus,
anthrax toxin of Bacillus anthracis, or Shiga-like toxin type I or
II of Escherichia coli.
6. A method for treating an infectious disease in a subject in need
thereof, comprising the step of administering to the subject an
effective amount of the molecular construct according to claim
1.
7. The method of claim 6, wherein the viral protein is F protein of
respiratory syncytia virus (RSV), gp120 protein of human deficiency
virus type 1 (HIV-1), hemagglutinin A (HA) protein of influenza A
virus, or glycoprotein of cytomegalovirus.
8. The method of claim 6, wherein the bacterial protein is
endotoxin of Gram(-) bacteria, surface antigen of Clostridium
difficile, lipoteichoic acid of staphylococcus aureus, anthrax
toxin of Bacillus anthracis, or Shiga-like toxin type I or II of
Escherichia coli.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] 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.
[0003] 2. Description of the Related Art
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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. 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
[0011] 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.
[0012] <I> Molecular Construct for Treating Central Nervous
System Diseases and Uses Thereof
[0013] In this 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. Targeting and effector elements of the present
Fc-based molecular constructs are specifically selected such that
these Fc-based molecular constructs are suitable for use in the
treatment of central nervous system (CNS) diseases, or for use in
the manufacture of a medicament for treating CNS diseases. As could
be appreciated, methods for treating CNS diseases using such
Fc-based molecular constructs also fall within the aspect of the
present disclosure.
[0014] 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 pair of effector elements, and a pair of
targeting elements. The pair of effector element is interferon
.beta.1a (INF-.beta.1a) or INF-.beta.1b, or an antibody fragment
specific for integrin .alpha.4 or .beta.-amyloid, while the pair
targeting elements is an antibody fragment specific for human
transferrin receptor or human insulin receptor.
[0015] In the case where the pair of effector elements is linked to
the N-termini of the pair of CH2-CH3 segments, the pair of
targeting elements is linked to the C-termini of the pair of
CH2-CH3 segments, and vice versa. Alternatively, when the pair of
effectors elements and the pair of targeting elements is both in
the form of single-chain variable fragments (scFvs), then the pair
of targeting elements is linked to the N-termini of the 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.
[0016] 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.
[0017] In some examples, the pair of effector elements or the 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.sub.K
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.
[0018] According to certain optional embodiments, the effector
element is INF-.beta.1a, INF-.beta.1b, or an scFv specific for
integrin .alpha.4, while the targeting element is an scFv specific
for human transferrin receptor. In particular, this molecular
construct is suitable for treating multiple sclerosis.
[0019] According to other optional embodiments, the effector
element is an scFv specific for .beta.-amyloid, while the targeting
element is an scFv specific for human transferrin receptor. In
particular, this molecular construct is suitable for treating
Alzheimer's disease.
[0020] Methods for treating CNS diseases in a subject in need
thereof comprise the step of administering to the subject an
effective amount of the molecular construct of this aspect. CNS
diseases treatable by this method include multiple sclerosis and
Alzheimer's disease.
[0021] <II > Molecular Construct for Treating Infectious
Diseases
[0022] In this 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. Targeting and effector elements of the present
Fc-based molecular constructs are specifically selected such that
these Fc-based molecular constructs are suitable for use in the
treatment of diseases/conditions associated with viral or bacterial
infection, or for use in the manufacture of a medicament for
treating such diseases/conditions. As could be appreciated, methods
for treating diseases/conditions associated with viral or bacterial
infection using such Fc-based molecular constructs also fall within
the aspect of the present disclosure.
[0023] 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 pair of effector elements, and a pair of
targeting elements. The pair of effector element is an antibody
fragment specific for CD32 or CD16b, while the pair targeting
elements is an antibody fragment specific for a viral protein or a
bacterial protein.
[0024] In the case where the pair of effector elements is linked to
the N-termini of the pair of CH2-CH3 segments, the pair of
targeting elements is linked to the C-termini of the pair of
CH2-CH3 segments, and vice versa. Alternatively, when the pair of
effectors elements and the pair of targeting elements is both in
the form of single-chain variable fragments (scFvs), then the pair
of targeting elements is linked to the N-termini of the 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.
[0025] 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.
[0026] In some examples, the pair of effector elements or the 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.sub.K
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.
[0027] According to certain optional embodiments, the effector
element is an scFv specific for CD32 or CD16b, while the targeting
element is an scFv specific for a viral protein. For example, the
viral protein can be F protein of respiratory syncytia virus (RSV),
gp120 protein of human immunodeficiency virus type 1 (HIV-1),
hemagglutinin A (HA) protein of influenza A virus, or glycoprotein
of cytomegalovirus. In particular, this molecular construct is
suitable for treating viral infecitons.
[0028] According to other optional embodiments, the effector
element is an scFv specific for Cd35 or C16b, while the targeting
element is an scFv specific for a bacterial protein. Examples of
the bacterial protein include, but are not limited to, the
endotoxin of Gram(-) bacteria, the surface antigen of Clostridium
difficile, the lipoteichoic acid of Saphylococcus aureus, the
anthrax toxin of Bacillus anthracis, or the Shiga-like toxin type I
or II of Escherichia coli. In particular, such molecular construct
is suitable for treating bacterial infecitons.
[0029] Methods for treating diseases/conditions associated with
infections (e.g., viral or bacterial infections) in a subject in
need thereof comprise the step of administering to the subject an
effective amount of the molecular construct of this aspect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The present description will be better understood from the
following detailed description read in light of the accompanying
drawings briefly discussed below.
[0031] FIGS. 1A to 1C are schematic diagrams illustrating Fc-based
molecular constructs according to various embodiments of the
present disclosure.
[0032] FIG. 2 is a schematic diagram illustrating an Fc-based
molecular construct according to various embodiments of the present
disclosure.
[0033] FIGS. 3A and 3B are schematic diagrams illustrating an
Fc-based molecular constructs according to various embodiments of
the present disclosure.
[0034] FIG. 4A shows the SDS-PAGE analysis result of purified
recombinant 2-chain (scFv .alpha. RSV)-hIgG1.Fc-(scFv .alpha. CD32)
fusion protein; FIG. 4B and FIG. 4C provide the results of ELISA
analyses that respectively illustrate the binding activities of the
purified recombinant fusion protein of FIG. 4A to Protein F of RSV
(FIG. 30B) and to ectodomain of CD32a (FIG. 30C).
[0035] FIG. 5A shows the SDS-PAGE analysis result of the purified
recombinant 2-chain (scFv .alpha. endotoxin)-hIgG1.Fc-(scFv .alpha.
CD32) fusion protein; and FIG. 5B and FIG. 31C provides the results
of ELISA analyses that respectively illustrate the binding affinity
of the purified recombinant fusion protein of FIG. 5A to endotoxin
(FIG. 5B) and to ectodomain of CD32a (FIG. 5C).
[0036] FIG. 6A and FIG. 6B respectively show the SDS-PAGE analysis
result and the ELISA results of the purified recombinant 2-chain
(Interferon-.beta.-1a)-hIgG4.Fc-(scFv .alpha. TfR1) fusion
protein.
[0037] FIG. 7A and FIG. 7B respectively shows the SDS-PAGE analysis
result and staining result of the purified recombinant 2-chain
(scFv .alpha. integrin .alpha.4)-hIgG4.Fc-(scFv .alpha. TfR1)
fusion protein.
[0038] FIG. 8 shows the ELISA analysis result of the effect of the
purified recombinant 2-chain (scFv .alpha.
endotoxin)-hIgG1.Fc-(scFv .alpha. CD32a) fusion protein on
inhibiting TNF-.alpha. secretion.
[0039] 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
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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,
phosphoramide, carbamate, hydroxylate, and the like.
[0050] 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.
[0051] 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.
[0052] "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##
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.
[0053] 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.
[0054] 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.
[0055] 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 any
of a protein, a polypeptide (including mutants or biologically
active fragments thereof), a polysaccharide, a glycoprotein, a
glycolipid, a nucleic acid, or a combination thereof.
[0056] 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.
[0057] 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.-8 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.
[0058] 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.
[0059] The term "effective amount" as used herein refers to the
quantity of the present molecular construct 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).
[0060] 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.
[0061] 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.
[0062] 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.
[0063] Diseases that can Benefit from Better Drug Targeting
[0064] 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. Certain antibody
drugs, which target infectious microorganisms or their toxic
products, can be improved, if they are empowered with the ability
to recruit immunocytes, which phagocytose and clear the
antibody-bound particles. Following are primary examples of
diseases, in which drugs can be improved if they can be
preferentially distributed to the disease sites or cells or if they
can recruit phagocytic immunocytes.
[0065] I Central Nervous System Diseases
[0066] For treating diseases of the central nervous system (CNS),
the therapeutic agents are often required to pass through the
blood-brain barrier (BBB) to get into the CNS. Some therapeutic
agents do not get into the CNS; they regulate certain activities,
such as immune activities, in the peripheral, which then modulates
the diseased conditions in the CNS. The BBB is formed by the
endothelial cells lining the capillaries of blood vessels in the
CNS. Unlike the capillaries in the peripheral tissues and organs,
the capillary endothelial cells in the BBB are connected by tight
junctions formed by occludin, claudins, and junctional adhesion
molecules.
[0067] At least six antibodies, namely, aducanumab, bapinerumab,
crenezumab, gantenerumab, ponezumab, and solanezumab, specific for
.beta.-amyloid, which is responsible for causing Alzheimer's
disease, have been developed and placed in clinical development.
These antibodies generally fall short of satisfactory therapeutic
efficacy in improving Alzheimer's disease. A general belief is that
if those antibodies are to achieve therapeutic efficacy, a
significant portion must get across the BBB to enter the injured
sites in the CNS. However, only very minute portions of those
antibodies get across the BBB.
[0068] Interferon-.beta.-1a (IFN-.beta.-1a) and
interferon-.beta.-1b (IFN-.beta.-1b) have been used for the
treatment of multiple sclerosis (MS). The pharmaceuticals,
IFN-.beta.-1a produced by mammalian cells and IFN-.beta.-1b
produced in E. coli, are one-chain protein of 166 amino acid
residues containing one disulfide bond. It has been claimed that
those therapeutic agents reduce relapse of MS in 18-38% of treated
patients. The mechanisms of action of IFN-.beta.-1a and
IFN-.beta.-1b are very complex and not completely understood,
involving the increased generation of anti-inflammatory immune
cells and factors and the down-regulation of pro-inflammatory cells
and factors. IFN-.beta. treatment in MS patients also reduces the
trafficking of pro-inflammatory T cells across the BBB. It is yet
unanswered whether IFN-.beta.-1a and IFN-.beta.-1b mediate their
pharmacologic effects in part by getting into the injured sites in
the CNS.
[0069] When an antibody or protein therapeutic is administered in
the body's peripheral, only a very minute amount (about 0.1%)
reaches to the CNS, because only a very minute portion of the
protein therapeutic gets across the BBB. However, it has also been
found that in many diseases of the CNS, including Alzheimer's
disease and multiple sclerosis, the inflammation at the diseased
sites renders the BBB to breakdown, leading to increased
permeability. Therefore, we rationalize that if a larger proportion
of an administered antibody specific for .beta.-amyloid or
IFN-.beta.-1a and IFN-.beta.-1b is channeled to the BBB, a higher
percentage of the therapeutic agents can pass through the BBB and
better therapeutic effects can be achieved.
[0070] Furthermore, some therapeutic agents have been developed to
inhibit the entry of inflammatory immunocytes to across the BBB. A
notable example is natalizumab specific for the cell adhesion
molecule integrin .alpha.4. The antibody functions by inhibiting
inflammatory immune cells to attach to and pass through the
epithelial layer lining the BBB. While natalizumab has been shown
to be therapeutic efficacious, it has serious immunosuppressive
side effect. In particular, it causes progressive multifocal
leukoencephalopathy, an opportunistic infection caused by John
Cunningham virus (JC virus). We therefor rationalize that if a
larger proportion of an antibody specific for integrin .alpha.4 is
recruited to the BBB, a smaller dose will be required, better
therapeutic effects can be achieved, and fewer side effects will
occur.
[0071] The endothelial cells in the capillaries forming the BBB
express transferrin receptors and insulin receptors, which mediate
the transcytosis of transferrin and insulin molecules,
respectively, to the cerebral parenchyma. For using the transferrin
receptor as a ferry, only a small proportion gets through while the
reaming bulk are trapped or degraded. Because the endothelial cells
lining the capillaries in other parts of the vasculature do not
express transferrin receptors, the transferrin receptors on the
endothelial cells in the BBB can serve as site-specific antigen for
recruiting administered therapeutics. Once the therapeutic is
concentrated in the BBB, an increased proportion of it will pass
through the capillaries.
[0072] We also rationalize that when the mechanisms for channeling
pharmaceuticals to the BBB is established, anti-inflammatory drugs,
such as anti-TNF-.alpha., anti-IL12/IL-23, anti-IL17, and anti-CD3,
should be investigated for their therapeutic effects on many types
of diseases of the CNS.
[0073] For the antibody therapeutic specific for integrin .alpha.4,
the transferrin receptor is used as a target site recruiter. For
Alzheimer disease, the effector moiety can be a few copies of scFv
specific for 3-amyloid; for treating multiple sclerosis, the
effector moiety can be a few copies of IFN-.beta.-1a or
IFN-.beta.-1b, or a few copies of scFv specific for integrin
.alpha.4.
[0074] Embodiments of the present disclosure disclose several T-E
molecules respectively exist in single multi-arm linker-units or
joint-linker configurations, each contains scFv specific for
transferrin receptor as the targeting element and IFN-.beta.-1a or
IFN-.beta.-1b or scFv specific for integrin .alpha.-4 as the
effector element. Alternative embodiments disclose T-E molecules
respectively exist in single linker-units or joint-linker
configurations, each contains scFv specific for transferrin
receptor as the targeting element and scFv specific for
.beta.-amyloid as the effector element.
[0075] Fingolimod is an immunosuppressive drug that is derived from
a natural product myriocin originally isolated from certain fungi.
Fingolimod has been approved for reducing the relapse of
relapsing-remitting multiple sclerosis. Fingolimod is
phosphorylated in vivo to form fingolimod-phosphate, which
resembles naturally occurring sphingosine-1-phosphate (S1P), an
extracellular lipid mediator, and can bind to 4 of the 5 S1P
receptors. The S1P receptors are expressed on lymphocytes and
involved in lymphocyte migration. A generally pharmacologic
mechanism of fingolimod is that it inhibits lymphocytes egress from
the lymphoid tissues to the circulation and hence to the CNS.
Fingolimod can cross BBB to enter CNS and many cell types in the
CNS express S1P receptors, which play roles in cell proliferation,
morphology, and migration. It is believed that fingolimod can have
direct on the CNS. The administration of fingolimod causes common
side effects of headache and fatigue, and severe side effects of
skin cancer, macular edema, and fatal infections, such as
hemorrhaging focal encephalitis.
[0076] A fingolimod molecule has an NH2 group and thus provides a
functional group to couple with a bi-functional linker with an NHS
group. One preferred embodiment of the present invention is to
prepare a T-E construct, which contains a targeting element for
delivery to the BBB and a drug bundle of fingolimod as an effector
element. For a bundle of fingolimod, 5-10 molecules are
incorporated to a linker unit, using either a cleavable linker or
non-cleavable linker to conjugate fingolimod molecules to the
linking arms of a linker unit. Since fingolimod, after uptake in a
patient, is modified to fingolimod phosphate to resemble
sphingosine1-phosphate and become active, the drug bundle is
alternatively prepared with fingolimod phosphate. A linker unit
with fingolimod or fingolimod phosphate bundle is conjugated with 1
or 2 scFv specific for a transferrin receptor I. Upon
administration of the molecular construct, a portion of it is
carried to the BBB. The fingolimod molecules released from the
cleavable linkers pass through the BBB and enter the CNS. Or, a
portion of the entire construct enters the CNS. Cleavable linkers
can be designed by employing a number of cleaving mechanisms. An
installment of S--S bond is often used, since S--S disulfide bond
can be cleaved by a reduction reaction at the target tissue site. A
peptide bond between amino acids, which is sensitive to proteases,
such as matrix metalloproteinases in many tissues and cathepsins in
endosomes in target cells, is also commonly used as a cleavable
bond in many linker designs.
[0077] II Infectious Disease
[0078] Although large numbers of monoclonal antibodies have been
made against components of a various viruses, bacteria, and fungi,
which cause serious infectious in humans and animals, few
monoclonal antibodies have been developed into preventive
treatments or therapeutic agents to counter infections. These
shortcomings can be attributed to a few major factors. One major
factor is the infectious microorganisms and their products have
different serotypes and variable reactivity toward a particular
antibody. Another reason is that the targeted microorganisms
undergo mutations and escape the targeting of a particular
antibody.
[0079] The T-E molecular design of the present invention can also
be applied for the prevention and treatment of infectious diseases.
The plurality of the linking arms can enhance the avidity and
specificity of binding to target infectious microorganisms or their
products and elicit immune functions to facilitate the clearance of
the microorganisms and their products. We reason that the avidity
enhancement and the recruitment of immune clearance function can
somehow overcome the stereotypic difference and mutational
problems. Such improvements should increase the efficacy of the
candidate antibodies for the prevention and therapy of infectious
diseases. Many antibodies, which have failed to meet expectation in
clinical trials, may be configured with the present invention and
re-investigated.
[0080] A preferred set of embodiment of the present invention is to
employ joint-linkers configuration with one linker-unit for
targeting and one linker-unit for recruiting effector function. An
alternative set of preferred embodiment is to employ single
linker-units with multiple linking arms for targeting elements and
a coupling arm for an effector element. The targeting elements may
be one of the two categories: (1) scFv or sdAb specific for a
surface component of a microorganism or its product, e.g., envelope
protein gp120 of human immunodeficiency virus type 1 (HIV-1), F
protein of respiratory syncytia virus (RSV), a surface antigen of
Clostridium difficile or Staphylococcus aureus, or endotoxin of
Gram-negative bacteria or Shiga-like toxin of Escherichia coli, or
(2) the extracellular portions of cell surface receptors of
viruses, such as the HIV-1 gp120-binding CD4 domain.
[0081] The effector elements are 1 or 2 scFv or sdAb specific for
one Fc receptor of IgG, e.g. Fc.gamma.RIIA (CD32), Fc.gamma.RIIIB
(CD16b), or Fc.gamma.RI (CD64). Those receptors are expressed on
neutrophils, macrophages, and eosinophils and are the key molecules
mediating phagocytosis of antibody-bound microorganisms.
Fc.gamma.RIIA and Fc.gamma.RIIIB bind to IgG with low affinity (Kd
in the range of 10.sup.-6 to 10.sup.-7), and Fc.gamma.RI binds to
IgG1 and IgG3 with high affinity (Kd 10.sup.-9). It is advantageous
to employ scFv or sdAb specific for Fc.gamma.RIIA or
Fc.gamma.RIIIB, because they can compete favorably with IgG in
binding to the receptors.
[0082] The antibodies specific for carbohydrate antigens on
bacterial surface are usually weak in binding affinity and are
expressed in IgM rather than IgG. An IgM molecule has 10 Fv's
(antigen-binding sites). However, an IgM molecule, which has a
molecular weight of about 1000 kd, cannot cross capillaries and
reach to extravascular space. With the configuration of the present
invention, a molecular construct carrying 6 scFv or 10 sdAb will
have a molecular weight of about 150 kd.
[0083] In employing antibody-based therapeutics for clearing
viruses, it is important that the therapeutic does not lead to
FcR-mediated enhancement of viral infection. In those cases, the
bound viral particles are not phagocytosed and digested. Some
viruses, such as Dengue virus can multiply in phagocytes. Thus, if
the viral particles gain access to a cell and enter the bound cells
without being destroyed, the virus can multiply in the infected
cells. Therefore, a set of preferred embodiments of this invention
is that the molecular construct contains 2 or more scFv specific
for an Fc.gamma. receptor and can bind to multiple Fc.gamma.
receptor molecules on phagocyte cell surface, so that the bound
viral particles are destined to phagocytosis pathway.
[0084] Among the many antibodies specific for viruses, bacteria, or
their products, which have been in clinical trials, only antibodies
specific for RSV have been approved for clinical uses. Even for
antibodies against RSV, they are only approved for prevention, and
not for treatment of on-going infection. It is desirable that an
anti-RSV antibody can be developed for treating already-infected
subjects. The other antibodies are still in clinical development or
have failed in clinical trials. With the molecular construct
platforms of this invention, all of these antibodies can be
employed for improved efficacy. A partial list of those antibodies
are: [0085] (1) Palivizumab and felvizumab specific for RSV F
protein [0086] (2) Suvizumab specific for HIV-1 gp120 [0087] (3)
Libivirumab, exbivirumab, tuvirumab specific for hepatitis B
surface antigen (HBsAg) of HBV [0088] (4) CR6261 mAb, diridavumab,
and firivumab specific for hemagglutinin A of influenza A virus
[0089] (5) Regavirumab and sevirumab specific for glycoprotein of
cytomegalovirus [0090] (6) Rafivirumab specific for glycoprotein of
rabies virus [0091] (7) Actoxumab and bezlotoxumab specific for
surface antigen of Clostridium difficile [0092] (8) Obiltoxaximab
and raxibacumab specific for Bacillus anthracis anthrax [0093] (9)
Panobacumab (human IgM monoclonal antibody) specific for
Pseudomonas aeruginosa serotype IATS O11 [0094] (10) Tefibazumab
and tosatoxumab specific for clumping factor A of Staphylococcus
aureus [0095] (11) Edobacomab specific for endotoxin of
Gram-negative bacteria for treating sepsis [0096] (12) Pagibaximab
specific for lipoteichoic acid of staphylococcus areus for treating
staphylococcal sepsis [0097] (13) Raxibacumab (human monoclonal
antibody) specific anthrax toxin [0098] (14) Pritoxaximab,
setoxaximab, and urtoxazumab specific for Shiga-like toxin type I
or II of Escherichia coli.
[0099] Fc-Based Molecular Constructs for Treating Central Nervous
System Diseases or Infectious Diseases
[0100] 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 or any other antibody fragments). 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.
[0101] 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, they
should also be positioned at the terminus. If the effector elements
function by binding to and neutralizing soluble factors, they can
be positioned between a terminal targeting or effector element and
CH2-CH3.
[0102] By selecting the T-E elements of the present Fc-based
molecular construct, the molecular construct can be used to treat
central nervous system (CNS) diseases or infectious diseases. The
present disclosure is also advantageous in that, in some
embodiments, it utilizes the linker unit according to the first
aspect of the present disclosure, which provides a facile means for
controlling the number of the targeting and effector elements of
the present Fc-based molecular constructs. Depending on the
targeting and/or effector elements selected, the present Fc-based
molecular construct may take different configurations, which are
discussed below, respectively.
[0103] In the present Fc-based molecular constructs, both the
targeting element and effector element are antibodies or fragments
thereof.
[0104] 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 pair of effector elements E1 linked to the N-termini
of the CH2-CH3 chains 810, and a 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 scFvs.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] In some examples, the pair of effector elements or the 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.sub.K
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.
[0109] For example, in the Fc-based molecular construct 900 of FIG.
2, each of the two targeting elements T1 comprises the V.sub.H-CH1
domain 820 and the V.sub.L-C.sub.K 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 900
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.
[0110] According to some embodiments, the present Fc-based
molecular construct has an effector element which is a peptide.
[0111] For example, according to certain embodiments of the present
disclosure, the effector element can be a peptide with certain
therapeutic effect, while the targeting element is an antibody or a
fragment thereof (see, FIGS. 3A and 3B). As illustrated, the
Fc-based molecular construct 1000A of FIG. 3A comprises a pair of
targeting elements T1 (as scFvs) linked to the N-termini of the
pair of CH2-CH3 segments 1210, and a pair of effector elements E1
(in the form of therapeutic peptides) linked to the C-termini of
the pair of CH2-CH3 segments 1210.
[0112] Similarly, in the Fc-based molecular construct 1000B of FIG.
3B, the pair of targeting elements T1 (as scFvs) is linked to the
C-termini of the pair of CH2-CH3 segments 1210, whereas the pair of
effector elements E1 (in the form of therapeutic peptides) is
linked to the C-termini of the pair of CH2-CH3 segments 1210.
[0113] As could be appreciated, for Fc-base molecular constructs
that use a peptide as the effector element, the targeting element
can be constructed into a Fab fragment, so that the molecular
constructs take the IgG configuration.
[0114] In the configuration illustrated in FIGS. 1A to 3B, 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.
[0115] Functional Elements Suitable for Use with Fc-Based Molecular
Construct
[0116] Now that the basic structural arrangements of the 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] To treat central nervous system (CNS) diseases, an antibody
(or a fragment thereof) specific for transferrin receptor can be
used as the targeting element, in connection with effector elements
suitable for the particular CNS disease. For example, Fc-based
molecular constructs for the treatment of multiple sclerosis may
use an scFv specific for integrin-.alpha.4 as the effector element.
In the case of Alzheimer's disease, illustrative Fc-based molecular
constructs can use an scFv specific for .beta.-amyloid as the
effector element. The above-mentioned Fc-based molecular constructs
for treating CNS diseases may take the configuration described in
connection with any of FIGS. 1A to 1C, and FIG. 2.
[0118] Fc-based molecular constructs for treating multiple
sclerosis may also use INF-.beta.1a or INF-.beta.1b as the effector
elements. In this case, the Fc-based molecular constructs may take
the configuration described in connection with FIG. 3A or 3B.
[0119] In constructing Fc-based molecular constructs for treating
diseases/conditions associated with infection (such as viral
infections or bacterial infections), one may use an antibody (or a
fragment thereof) specific for a viral protein or bacterial protein
as the targeting element. As to the effector elements for treating
infections, an antibody (or a fragment thereof) specific for CD32
or CD16b can be used. These Fc-based molecular constructs may take
the configuration described in connection with any of FIGS. 1A to
1C, and FIG. 2.
[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] III-(iii) Use of Fc-Based Molecular Construct
[0122] The present disclosure also pertains to method for treating
CNS diseases using the suitable Fc-based molecular construct.
Generally, the method comprises the step of administering to a
subject in need of such treatment an effective amount of the
Fc-based molecular construct according to embodiments of the
present disclosure.
[0123] The present disclosure further pertains to method for
treating infections using the suitable Fc-based molecular
construct. Generally, the method comprises the step of
administering to a subject in need of such treatment an effective
amount of the Fc-based molecular construct according to embodiments
of the present disclosure.
EXPERIMENTAL EXAMPLES
Example 1
Construction of a Gene Segment Encoding 2-Chain IgG1.Fc Fusion
Protein Containing scFv Specific for Protein F of RSV and scFv
Specific for Ectodomain of CD32a
[0124] The scFv1-CH2-CH3-scFv2 (human .gamma.1) recombinant chain
was configured by fusing two scFvs, in which the first one specific
for Protein F of RSV fused to the N-terminal of CH2 domain of
IgG1.Fc through a flexible hinge region, while the second one
specific for ectodomain of CD32a was fused to the C-terminal of CH3
domain through a flexible linker, (GGGGS).sub.3.
[0125] Both of the scFvs had an orientation of
V.sub.L-linker-V.sub.H. The V.sub.L and V.sub.H in each of the two
scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.
The sequence of the recombinant chain in the IgG1.Fc fusion protein
molecular construct is shown as SEQ ID NO: 1.
[0126] Illustrated below is the configuration of the prepared
2-chain (scFv .alpha. RSV)-(scFv .alpha. CD32a)-hIgG1.Fc molecular
construct.
##STR00001##
Example 2
Expression and Purification of Recombinant 2-Chain (scFv .alpha.
RSV)-hIgG1.Fc-(scFv .alpha. CD32) Fusion Protein
[0127] 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.
At the time 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 7 days. Culture supernatants
were harvested and recombinant 2-chain (scFv .alpha.
RSV)-hIgG1.Fc-(scFv .alpha. CD32a) fusion protein in the media was
purified using Protein A chromatography. Following buffer exchange
to PBS, the concentration of (scFv .alpha. RSV)-hIgG1.Fc-(scFv
.alpha. CD32a) protein was determined and analyzed by 12% SDS-PAGE
shown in FIG. 4A. The Fc-fusion molecular construct was revealed as
the major band at about 85 kDa, consistent with the expected
size.
Example 3
ELISA Analysis of the Binding of Recombinant 2-Chain (scFv
.alpha.RSV)-hIgG1.Fc-(scFv .alpha. CD32a) Fusion Protein
[0128] To examine the binding ability of recombinant 2-chain (scFv
.alpha. RSV)-hIgG1.Fc-(scFv .alpha. CD32a) fusion protein to both
Protein F of RSV and ectodomain of CD32a, ELISA assay was
performed. ELISA plates were coated with 2 .mu.g/mL of Protein F of
RSV (Sino biological Inc.). Recombinant 2-chain (scFv .alpha.
RSV)-hIgG1.Fc-(scFv .alpha. CD32a) fusion protein and (scFv .alpha.
RSV)-hIgG1.Fc were detected by HRP-conjugated goat anti-human
IgG1.Fc. The ELISA results in FIG. 4B show that the recombinant
2-chain (scFv .alpha. RSV)-hIgG1.Fc-(scFv .alpha. CD32a) fusion
protein bind to Protein F of RSV, using adalimumab scFv as a
control scFv.
[0129] FIG. 4C shows binding activity of the recombinant Fc-fusion
protein to ectodomain of CD32a. ELISA plates were coated with 5
.mu.g/mL of recombinant ectodomain of CD32a. Recombinant 2-chain
(scFv .alpha. RSV)-hIgG1.Fc-(scFv .alpha. CD32a) fusion protein was
detected by HRP-conjugated goat anti-human IgG1.Fc. FIG. 4C shows
that the recombinant 2-chain (scFv .alpha. RSV)-hIgG1.Fc-(scFv
.alpha. CD32a) Fc-fusion protein has binding activity to
recombinant ectodomain of CD32a. Recombinant 2-chain (scFv .alpha.
endotoxin)-IgG1.Fc protein was used as a control antibody.
Example 4
Preparation of 2-Chain IgG1.Fc Fusion Protein Containing scFv
Specific for Endotoxin and scFv Specific for Ectodomain of
CD32a
[0130] The scFv1-CH2-CH3-scFv2 (human .gamma.1) recombinant chain
was configured by fusing two scFvs, in which the first one specific
for endotoxin was fused to the N-terminal of CH2 domain of IgG1.Fc
through a flexible hinge region, and the second one specific for
ectodomain was fused to the C-terminal of CH3 domain through a
flexible linker, (GGGGS).sub.3.
[0131] Both of the scFvs had an orientation of
V.sub.L-linker-V.sub.H. The V.sub.L and V.sub.H in each of the two
scFv were connected by a hydrophilic linker,
GSTSGSGKPGSGEGSTKG.
[0132] The sequence of the recombinant chain in the IgG1.Fc fusion
protein molecular construct is shown as SEQ ID NO: 2. The
expression of the constructed gene 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 SDA-PAGE results in FIG. 5A
shows that the recombinant chain of the new construct has a size of
about 85 kDa, consistent with the expected size.
[0133] FIG. 5B shows ELISA results of the recombinant 2-chain (scFv
.alpha. endotoxin)-(scFv .alpha. CD32a)-hIgG1.Fc binding to E.coli
LPS 0111:B4 (Sigma Aldrich). ELISA plates were coated with 50
.mu.g/ml poly-L-lysine. Subsequently, the poly-L-lysine-coated
plates were further coated with 10 .mu.g/ml E.coli LPS 0111:B4. The
recombinant fusion protein was detected by HRP-conjugated goat
anti-human IgG.Fc. The ELISA results show that the present
recombinant Fc-fusion protein has binding activity to E.coli LPS
0111:B4 (Sigma Aldrich); FIG. 5C shows that the recombinant
Fc-fusion protein has binding activity to ectodomain of CD32a.
[0134] Illustrated below is the configuration of the thus-prepared
2-chain (scFv .alpha. endotoxin)-(scFv .alpha. CD32)-hIgG1.Fc
molecular construct.
##STR00002##
Example 5
Construction of a Gene Segment Encoding 2-Chain IgG4.Fc Fusion
Protein Containing Interferon-.beta.-1a and scFv Specific for
Ectodomain of TfR1
[0135] The 2-chain IgG.Fc fusion protein was prepared by
configuring (interferon-.beta.-1a)-CH2-CH3-(scFv .alpha. TfR1)
(human .gamma.4) in a recombinant chain. The C-terminal of the
interferon-.beta.-1a was fused to the N-terminal of CH2 via a
linker, GGGGSGGGASGGS. The scFv specific for ectodomain of TfR1 was
fused to the C-terminal of CH3 domain through a flexible linker,
(GGGGS).sub.3.
[0136] The scFv (specific for ectodomain of TfR1) had an
orientation of V.sub.L-linker-V.sub.H. The V.sub.L and V.sub.H in
the scFv were connected by a hydrophilic linker,
GSTSGSGKPGSGEGSTKG. The sequence of the recombinant chain in the
IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO:
3.
##STR00003##
[0137] Illustrated herein is the configuration of the prepared
2-chain (interferon-.beta.-1a)-IgG4.Fc-(scFv .alpha. TfR1)
molecular construct.
Example 6
Expression and Purification of Recombinant 2-Chain
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) Fusion
Protein
[0138] 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.
At the time 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 7 days. Culture supernatants
were harvested and recombinant 2-chain
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) fusion protein
in the media was purified using Protein A chromatography. Following
buffer exchange to PBS, the concentration of
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) protein was
determined and analyzed by 8% SDS-PAGE shown in FIG. 6A. The
Fc-fusion molecular construct was revealed as the major band at
about 80 kDa, consistent with the expected size.
Example 7
Binding Analysis of Recombinant 2-Chain
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) Fusion Protein
Using ELISA and Flow Cytometry
[0139] Binding activity of recombinant
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) was assayed by
ELISA using a 96-well plate coated with recombinant
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) protein in 5
.mu.g/ml concentration, 100 .mu.l per well. The scFv specific for
ectodomain of TfR1 is as a negative control. Recombinant 2-chain
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) was detected by
HRP-conjugated rabbit anti-human interferon-.beta. polyclonal
antibody (Santa Cruz Biotechnology, Dallas, USA). Next, 50 .mu.l of
TMB substrate was added for color development. The reaction was
stopped by 50 .mu.l of 1M HCl. Absorbance at 450 nm was measured
with a plate reader, Each bar represents the mean OD450 value of
duplicate samples.
[0140] FIG. 6B shows ELISA analysis of the present the molecular
construct. The ELISA results show that
(Interferon-.beta.-1a)-hIgG1.Fc-(scFv .alpha. TfR1) fusion protein
bound specifically to recombinant ectodomain of TfR1 protein.
Example 8
Preparation of 2-Chain IgG4.Fc Fusion Protein Containing scFv
Specific for Integrin .alpha.4 and scFv Specific for Ectodomain of
TfR1
[0141] The V.sub.L and V.sub.H of the scFv specific for integrin
.alpha.4 were from monoclonal antibody natalizumab. The 2-chain
IgG.Fc fusion protein was prepared by configuring (scFv .alpha.
integrin .alpha.4)-CH2-CH3-(scFv .alpha. TfR1) (human .gamma.4) in
a recombinant chain. The C-terminal of the scFv specific for
integrin .alpha.4 was fused to the N-terminal of CH2 via a linker,
GGGGSGGGASGGS. The scFv specific for ectodomain of TfR1 was fused
to the C-terminal of CH3 domain through a flexible linker,
(GGGGS).sub.3. The result of 8% SDA-PAGE in FIG. 7A shows that the
recombinant chain of the new construct has a size of about 85 kDa
(indicated by arrow), consistent with the expected size.
[0142] The two scFv had the orientation of V.sub.L-linker-V.sub.H.
The V.sub.L and V.sub.H in each of the two scFv were connected by a
hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the
recombinant chain in the IgG4.Fc fusion protein molecular construct
is shown as SEQ ID NO: 4.
##STR00004##
[0143] Illustrated herein is the configuration of the prepared
2-chain (scFv .alpha. integrin .alpha.4)-IgG4.Fc-(scFv .alpha.
TfR1) molecular construct.
[0144] To examine the binding ability of recombinant 2-chain (scFv
.alpha. integrin .alpha.4)-IgG4.Fc-(scFv .alpha. TfR1) protein to
integrin .alpha.4-expressing Jurkat T cells, cell-binding assay was
performed by flow cytometry.
[0145] 1.times.10.sup.6 Jurkat T cells was maintained in the
RPMI1640 medium supplemented with 10% FBS at a density of
1.times.10.sup.5. The cells were kept in 37.degree. C. with 5%
CO.sub.2 in a humidified chamber. 1.times.10.sup.6 Jurkat T cells
were washed with the binding buffer (phosphate-buffered saline with
0.1% FBS, 2 mM EDTA and 20 ng/ml NaN.sub.3) twice. 10 .mu.g/ml of
Human BD Fc Block.TM. (BD Biosciences, San Jose, US) was added to
the washed Jurkat T cells to block Fc receptor mediated. Cells were
washed and incubated with 10 .mu.g/ml of recombinant (scFv a
integrin .alpha.4)-IgG4.Fc-(scFv .alpha. TfR1) protein on ice for
15 minutes, using recombinant 2-chain
(interferon-.beta.-1a)-IgG4.Fc-(scFv .alpha. TfR1) as a negative
control. Cells were washed again and incubated with FITC-conjugated
goat anti-human IgG.Fc (Caltag, Buckingham, UK), diluted 1:200 in
blocking buffer, at on ice for 15 min in the dark. The stained
cells were analyzed on a FACSCanto II flow cytometer (BD
Biosciences).
[0146] FIG. 7B shows results of the cell staining analysis of
recombinant 2-chain (scFv .alpha. integrin .alpha.4)-IgG4.Fc-(scFv
.alpha. TfR1) protein on integrin (14-expressing Jurkat T cells.
The construct bound to Jurkat T cells substantially positively.
Example 9
Assay of Biological Activity of 2-Chain IgG1.Fc Containing scFv
Specific for Endotoxin and scFv Specific for Ectodomain of CD32a on
Macrophage-Like U937 Cells
[0147] To test the effects of recombinant 2-chain (scFv .alpha.
endotoxin)-hIgG1.Fc-(scFv .alpha. CD32a) fusion protein on
inhibiting TNF-.alpha. secretion, ELISA was to determine the amount
of secreted TNF-.alpha. in the supernatant by macrophage-like U937
cells.
[0148] U937 cells were maintained in RPMI1640 supplemented with 10%
fetal bovine serum (Gibco) and 100 U/ml penicillin-streptomycin
(Gibco), at the density between 3.times.10.sup.5 and
2.times.10.sup.6 cells/ml. The cells were kept in 37.degree. C.
with 5% CO.sub.2 in a humidified chamber. To differentiate U937
into macrophage-like cells, 1.times.10.sup.6 cells/ml of U937 were
incubated with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA,
Sigma Aldrich). After 48 hours, non-adherent cells were removed,
and adherent cells were washed and seeded into 96-well plates.
[0149] 5.times.10.sup.4 cells/well of differentiated 0937 were
seeded into 96-well plates the day before assay. Cells were
stimulated with 1 .mu.g/ml E. coli LPS 0111:B4 (Sigma Aldrich)
alone, or premixes of LPS and 10 .mu.g/ml of (scFv .alpha.
endotoxin)-hIgG1Fc, 15 .mu.g/ml of (scFv .alpha. endotoxin)-hIgG1
Fc-(scFv .alpha. CD32a) or 2.5 .mu.g/ml of anti-CD32a scFv. The
stimulation proceeded for 2 hours before the supernatant was
collected. TNF-.alpha. production was measured by commercially
available ELISA kit (Biolegend).
[0150] TNF-.alpha. levels in U937 supernatant were measured using
an ELISA kit from R&D Systems. The wells of ELISA plates
(Greiner Bio-One) were coated with 4 .mu.g/mL of capture antibody
in PBS at 4.degree. C. overnight. Wells were subsequently blocked
by 0.5% in PBS for 1 hour and incubated with diluted culture
supernatant for 2 hours. 400 ng/mL of biotin-labeled detection
antibody was used followed by Streptavidin-HRP to detect bound
TNF-.alpha.. Chromogenic reaction was carried out using TMB
substrate (Clinical Science Products), and stopped by adding 1N
HCl. Plates were read at 450 nm absorbance. Concentrations of
TNF-.alpha. were determined by extrapolation from four-parameter
logistic fit standard curves generated from dilutions of standard
protein supplied by the manufacturer.
[0151] FIG. 8 shows that recombinant 2-chain (scFv .alpha.
endotoxin)-hIgG1 Fc-(scFv .alpha. CD32a) significantly reduced
TNF-.alpha. secretion stimulated by E. coli LPS 0111:B4, compared
to control antibodies 2-chain (scFv .alpha. endotoxin)-hIgG1 Fc
protein and anti-CD32a scFv or medium alone.
[0152] 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
41737PRTArtificial Sequence(anti-RSV scfv)-IgG1.Fc-(anti-CD32 scFv)
1Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys
Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Thr 115 120 125 Leu
Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr 130 135
140 Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Ser
145 150 155 160 Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
Trp Leu Ala 165 170 175 Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn
Pro Ser Leu Lys Ser 180 185 190 Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val Val Leu Lys 195 200 205 Val Thr Asn Met Asp Pro Ala
Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 210 215 220 Ser Met Ile Thr Asn
Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 225 230 235 240 Val Thr
Val Ser Ser Ala Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly 245 250 255
Gly Gly Gly Ser Pro Ala Pro Glu Leu 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 His Glu Asp
Pro Glu Val 290 295 300 Lys 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 Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val 325 330 335 Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Asp Tyr Lys Cys 340 345 350 Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 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 Arg Asp Glu Leu Thr Arg 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 Lys Leu Thr
Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln 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 Pro Gly Lys Gly Gly 465 470 475 480 Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln 485 490 495 Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 500 505
510 Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Ser Ala Leu Ala Trp
515 520 525 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Asp Ala 530 535 540 Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser 545 550 555 560 Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe 565 570 575 Ala Thr Tyr Tyr Cys Gln Gln
Phe Asn Ser Tyr Pro His Thr Phe Gly 580 585 590 Gln Gly Thr Lys Leu
Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly 595 600 605 Lys Pro Gly
Ser Gly Glu Gly Ser Thr Lys Gly Gln Val His Leu Val 610 615 620 Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser 625 630
635 640 Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp
Val 645 650 655 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val
Ile Trp Tyr 660 665 670 Asp Gly Ser Asn Tyr Tyr Tyr Thr Asp Ser Val
Lys Gly Arg Phe Thr 675 680 685 Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser 690 695 700 Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Asp Leu Gly 705 710 715 720 Ala Ala Ala Ser
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 725 730 735 Ser
2738PRTArtificial Sequence(anti-endotoxin scfv)-IgG1.Fc-(anti-CD32
scFv) 2Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu
Gly 1 5 10 15 Asp Thr Ile Ser Ile Thr Cys Arg Ala Ser Gln Asn Ile
Asn Ile Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Val
Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Ile Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr
Tyr Tyr Cys Leu Gln Gly Gln Ser Tyr Pro Arg 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 Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
130 135 140 Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
Tyr Met 145 150 155 160 Thr Trp Val Arg Gln Ala Pro Gly Lys Ala Pro
Glu Trp Leu Ala Leu 165 170 175 Ile Arg Asn Lys Arg Asn Gly Asp Thr
Ala Glu Tyr Ser Ala Ser Val 180 185 190 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Tyr Ser Arg Ser Ile Leu His 195 200 205 Leu Gln Met Asn Ala
Leu Arg Thr Glu Asp Ser Ala Thr Tyr Tyr Cys 210 215 220 Val Arg Gln
Gly Arg Gly Tyr Thr Leu Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240
Ser Val Thr Val Ser Ser Ala Ser Gly Gly Ser Gly Gly Gly Gly Ser 245
250 255 Gly Gly Gly Gly Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val 260 265 270 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 275 280 285 Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu 290 295 300 Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys 305 310 315 320 Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 325 330 335 Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Asp Tyr Lys 340 345 350 Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 355 360 365
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 370
375 380 Pro Ser Arg Asp Glu Leu Thr Arg Asn Gln Val Ser Leu Thr Cys
Leu 385 390 395 400 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 405 410 415 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 420 425 430 Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg 435 440 445 Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu 450 455 460 His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 465 470 475 480 Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile 485 490
495 Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
500 505 510 Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Ser Ala
Leu Ala 515 520 525 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr Asp 530 535 540 Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly 545 550 555 560 Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp 565 570 575 Phe Ala Thr Tyr Tyr
Cys Gln Gln Phe Asn Ser Tyr Pro His Thr Phe 580 585 590 Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Gly Ser Thr Ser Gly Ser 595 600 605 Gly
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val His Leu 610 615
620 Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu
625 630 635 640 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly
Met His Trp 645 650 655 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val Ala Val Ile Trp 660 665 670 Tyr Asp Gly Ser Asn Tyr Tyr Tyr Thr
Asp Ser Val Lys Gly Arg Phe 675 680 685 Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu Gln Met Asn 690 695 700 Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Leu 705 710 715 720 Gly Ala
Ala Ala Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 725 730 735
Ser Ser 3658PRTArtificial
Sequence(IFN-beta)Avonex-IgG4.Fc-(anti-TfR scFv) 3Met Ser Tyr Asn
Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln
Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30
Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35
40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu
Gln 50 55 60 Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr
Gly Trp Asn 65 70 75 80 Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val
Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr Val Leu Glu Glu Lys
Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys Leu Met Ser Ser
Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu His Tyr Leu
Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile Val Arg
Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160
Thr Gly Tyr Leu Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser 165
170 175 Gly Gly Ser Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu
Gly 180 185 190 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 195 200 205 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln 210 215 220 Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 225 230 235 240 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 245 250 255 Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 260 265 270 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 275 280 285
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 290
295 300 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser 305 310 315 320 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 325 330 335 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 340 345 350 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val 355 360 365 Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met 370 375 380 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 385 390 395 400 Leu
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 405 410
415 Gly Ser Asp Ile Val Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
420 425 430 Leu Gly Asp Thr Ile Leu Ile Thr Cys His Ala Ser Gln Asn
Ile Asn 435 440 445 Val Trp Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn
Ala Pro Lys Leu 450 455 460 Leu Ile Tyr Lys Ala Ser Asn Leu His Thr
Gly Val Pro Ser Arg Phe 465 470 475 480 Ser Gly Ser Gly Ser Gly Thr
Gly Phe Thr Leu Thr Ile Ser Ser Leu 485 490 495 Gln Pro Glu Asp Ile
Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr 500 505 510 Pro Trp Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ser 515 520 525 Thr
Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly 530 535
540 Gln Val Gln Leu Gln Gln Pro Gly Ala Ala Leu Val Arg Pro Gly Ala
545 550 555 560 Ser Met Arg Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Thr Tyr 565 570 575 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln
Gly Leu Glu Leu Ile 580 585 590 Gly Met Ile His Pro Ser Asp Ser Glu
Val Arg Leu Asn Gln Lys Phe 595 600 605 Lys Asp Lys Ala Thr Leu Thr
Val Asp Thr Ser Ser Ser Thr Ala Tyr 610 615 620 Met Gln Leu Asn Ser
Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 625 630 635 640 Ala Arg
Phe Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 645 650 655
Ser Ser 4738PRTArtificial Sequence(anti-integrin a4
scfv)--IgG4.Fc-(anti-TfR scFv) 4Ser Ile Val Met Thr Gln Thr Pro Lys
Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala
Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser
Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70
75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser 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 Val Lys 115 120 125 Leu Gln Gln Ser Gly Ala Glu Leu
Val Lys Pro Gly Ala Ser Val Lys 130 135 140 Leu Phe Cys Thr Ala Ser
Gly Phe Asn Ile Lys Asp Thr Tyr Met His 145 150 155 160 Trp Val Lys
Gln Arg Pro Gln Gln Gly Leu Glu Trp Ile Gly Arg Ile 165 170 175 Asp
Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe Gln Val Lys 180 185
190 Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Trp Leu Gln Leu
195 200 205 Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Asp Gly 210 215 220 Met Trp Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp
Gly Gln Gly Thr 225 230 235 240 Thr Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Ala Ser 245 250 255 Gly Gly Ser Pro Pro Cys Pro
Ser Cys Pro Ala Pro Glu Phe Leu Gly 260 265 270 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 290 295 300 Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 305 310
315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile 355 360 365 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser 385 390 395 400 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410 415 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 420 425 430
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 435
440 445 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 465 470 475 480 Leu Gly Lys Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly 485 490 495 Gly Ser Asp Ile Val Ile Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser 500 505 510 Leu Gly Asp Thr Ile Leu
Ile Thr Cys His Ala Ser Gln Asn Ile Asn 515 520 525 Val Trp Leu Ser
Trp Phe Gln Gln Lys Pro Gly Asn Ala Pro Lys Leu 530 535 540 Leu Ile
Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe 545 550 555
560 Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu
565 570 575 Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln
Ser Tyr 580 585 590 Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Gly Ser 595 600 605 Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly
Glu Gly Ser Thr Lys Gly 610 615 620 Gln Val Gln Leu Gln Gln Pro Gly
Ala Ala Leu Val Arg Pro Gly Ala 625 630 635 640 Ser Met Arg Leu Ser
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr 645 650 655 Trp Met Asn
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Leu Ile 660 665 670 Gly
Met Ile His Pro Ser Asp Ser Glu Val Arg Leu Asn Gln Lys Phe 675 680
685 Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
690 695 700 Met Gln Leu Asn Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 705 710 715 720 Ala Arg Phe Gly Leu Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr Val 725 730 735 Ser Ser
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