U.S. patent application number 15/114474 was filed with the patent office on 2016-12-01 for mhc class i epitope delivering polypeptides and cell-targeted molecules for direct cell killing and immune stimulation via mhc class i presentation and methods regarding the same.
This patent application is currently assigned to Molecular Templates, Inc.. The applicant listed for this patent is MOLECULAR TEMPLATES, INC.. Invention is credited to Eric Poma, Erin Willert.
Application Number | 20160347798 15/114474 |
Document ID | / |
Family ID | 52469328 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160347798 |
Kind Code |
A1 |
Poma; Eric ; et al. |
December 1, 2016 |
MHC CLASS I EPITOPE DELIVERING POLYPEPTIDES AND CELL-TARGETED
MOLECULES FOR DIRECT CELL KILLING AND IMMUNE STIMULATION VIA MHC
CLASS I PRESENTATION AND METHODS REGARDING THE SAME
Abstract
The present invention is directed to T-cell epitope delivering
polypeptides which deliver one or more CD8+ T-cell epitopes to the
MHC class I presentation pathway of a cell, including toxin-derived
polypeptides which comprise embedded T-cell epitopes and are
de-immunized. The present invention provides cell-targeted, CD8+
T-cell epitope delivering molecules for the targeted delivery of
cytotoxicity to certain cells, e.g., infected or malignant cells,
for the targeted killing of specific cell types, and the treatment
of a variety of diseases, disorders, and conditions, including
cancers, immune disorders, and microbial infections. The present
invention also provides methods of generating polypeptides capable
of delivering one or more heterologous T-cell epitopes to the MHC
class I presentation pathway, including polypeptides which are 1)
B-cell and/or CD4+ T-cell de-immunized, 2) comprise embedded T-cell
epitopes, and/or 3) comprises toxin effectors which retain toxin
functions.
Inventors: |
Poma; Eric; (New York,
NY) ; Willert; Erin; (Round Rock, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MOLECULAR TEMPLATES, INC. |
Georgetowm |
TX |
US |
|
|
Assignee: |
Molecular Templates, Inc.
Georgetown
TX
|
Family ID: |
52469328 |
Appl. No.: |
15/114474 |
Filed: |
January 26, 2015 |
PCT Filed: |
January 26, 2015 |
PCT NO: |
PCT/US2015/012968 |
371 Date: |
July 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61932000 |
Jan 27, 2014 |
|
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62049325 |
Sep 11, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61K 2039/6037 20130101; C12Y 302/02022 20130101; C07K 16/2866
20130101; A61P 37/02 20180101; C07K 16/2863 20130101; Y02A 50/489
20180101; C12N 9/1077 20130101; A61P 31/00 20180101; A61P 37/00
20180101; A61P 43/00 20180101; C07K 16/286 20130101; A61P 3/10
20180101; C07K 16/088 20130101; A61P 17/00 20180101; C12Y 204/02036
20130101; A61P 37/04 20180101; C07K 2319/55 20130101; A61P 1/04
20180101; A61P 5/14 20180101; A61P 35/00 20180101; C07K 16/00
20130101; C12N 9/2497 20130101; C07K 2319/33 20130101; A61P 29/00
20180101; C07K 2319/04 20130101; A61P 25/00 20180101; C07K 14/245
20130101; C07K 16/085 20130101; C07K 16/32 20130101; C07K 16/1063
20130101; Y02A 50/30 20180101; A61P 17/06 20180101; A61P 31/04
20180101; C07K 16/2887 20130101; C07K 14/25 20130101; C12N 15/62
20130101; A61P 31/18 20180101; A61P 37/06 20180101; C07K 2319/40
20130101; A61P 11/06 20180101; A61P 9/00 20180101 |
International
Class: |
C07K 14/25 20060101
C07K014/25; C12N 9/10 20060101 C12N009/10; C07K 14/245 20060101
C07K014/245 |
Claims
1-71. (canceled)
106. A polypeptide comprising an embedded, heterologous, CD8+
T-cell epitope, wherein the polypeptide is capable of intracellular
delivery of the T-cell epitope from an early endosomal compartment
to a MHC class I molecule of a cell in which the polypeptide is
present; and wherein the embedded, heterologous CD8+ T-cell epitope
replaces an equivalent number of amino acid residues in a parental
polypeptide such that the polypeptide comprising the epitope has
the same total number of amino acids as the parental
polypeptide.
107. The polypeptide of claim 106, comprising a proteasome
delivering effector polypeptide.
108. The polypeptide of claim 106, comprising a toxin effector
polypeptide capable of exhibiting one or more toxin effector
functions.
109. The polypeptide of claim 108, wherein the toxin effector
polypeptide comprises a proteasome delivering effector
polypeptide.
110. The polypeptide of claim 108 or claim 109, wherein the
heterologous, CD8+ T-cell epitope is embedded in the toxin effector
polypeptide.
111. The polypeptide of claim 110, wherein the toxin effector
polypeptide is capable of exhibiting one or more toxin effector
functions in addition to intracellular delivery of a CD8+ T-cell
epitope from an early endosomal compartment to a MHC class I
molecule of a cell in which the toxin effector polypeptide is
present.
112. The polypeptide of any one of claims 108-111, wherein the
toxin effector polypeptide is derived from a toxin selected from
the group consisting of: ABx toxin, ribosome inactivating protein
toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix
toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin,
mitogillin, pertussis toxin, pokeweed antiviral protein,
pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,
sarcin, Shiga toxin, and subtilase cytotoxin.
113. The polypeptide of claim 112, wherein the toxin effector
polypeptide is derived from a polypeptide selected from the group
of polypeptides represented by: (i) amino acids 75 to 251 of SEQ ID
NO:1, SEQ ID NO:2, or SEQ ID NO:3; (ii) amino acids 2 to 389 of SEQ
ID NO:45; (iii) amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2,
or SEQ ID NO:3; (iv) amino acids 1 to 251 of SEQ ID NO:1, SEQ ID
NO:2, or SEQ ID NO:3; and (v) amino acids 1 to 261 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3.
114. A polypeptide comprising an embedded, heterologous, CD8+
T-cell epitope disrupting an endogenous B-cell epitope and/or CD4+
T-cell epitope, wherein the embedded, heterologous CD8+ T-cell
epitope replaces an equivalent number of amino acid residues in a
parental polypeptide such that the polypeptide comprising the
epitope has the same total number of amino acids as the parental
polypeptide.
115. The polypeptide of claim 114, wherein the polypeptide is
capable of intracellular delivery of the CD8+ T-cell epitope to a
MHC class I molecule from an early endosomal compartment of a cell
in which the polypeptide is present.
116. The polypeptide of claim 114 or claim 115, comprising a toxin
effector polypeptide capable of exhibiting one or more toxin
effector functions.
117. The polypeptide of claim 116, wherein the heterologous, CD8+
T-cell epitope is embedded in the toxin effector polypeptide.
118. The polypeptide of claim 117, wherein the toxin effector
polypeptide is capable of exhibiting one or more toxin effector
functions in addition to intracellular delivery of a CD8+ T-cell
epitope from an early endosomal compartment to a MHC class I
molecule of a cell in which the toxin effector polypeptide is
present.
119. The polypeptide of claim 116, wherein the toxin effector
polypeptide is derived from a toxin selected from the group
consisting of: ABx toxin, ribosome inactivating protein toxin,
abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin,
claudin, diphtheria toxin, gelonin, heat-labile enterotoxin,
mitogillin, pertussis toxin, pokeweed antiviral protein,
pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,
sarcin, Shiga toxin, and subtilase cytotoxin.
120. The polypeptide of claim 119, wherein the toxin effector
polypeptide comprises a diphtheria toxin effector polypeptide
comprising amino acid sequences derived from the A and B Subunits
of at least one member of the diphtheria toxin family, wherein the
diphtheria toxin effector polypeptide comprises a disruption of at
least one B-cell epitope and/or CD4+ T-cell epitope region of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: 3-10 of SEQ ID NO:44, 15-31 of SEQ ID
NO:44, 32-54 of SEQ ID NO:44; 33-43 of SEQ ID NO:44, 71-77 of SEQ
ID NO:44, 93-113 of SEQ ID NO:44, 125-131 of SEQ ID NO:44, 138-146
of SEQ ID NO:44, 141-167 of SEQ ID NO:44, 165-175 of SEQ ID NO:44,
182-201 of SEQ ID NO:45, 185-191 of SEQ ID NO:44, and 225-238 of
SEQ ID NO:45; and wherein the diphtheria toxin effector polypeptide
is capable of routing to a cytosol compartment of a cell in which
the diphtheria toxin effector polypeptide is present.
121. The polypeptide of claim 119, wherein the diphtheria toxin
effector polypeptide is derived from the polypeptide represented by
amino acids 2 to 389 of SEQ ID NO:45.
122. The polypeptide of claim 119, wherein the toxin effector
polypeptide comprises a Shiga toxin effector polypeptide comprising
an amino acid sequence derived from an A Subunit of at least one
member of the Shiga toxin family, wherein the Shiga toxin effector
polypeptide comprises a disruption of at least one B-cell epitope
and/or CD4+ T-cell epitope region of the Shiga toxin A Subunit
amino acid sequence selected from the group of natively positioned
amino acids consisting of: the B-cell epitope regions: 1-15 of SEQ
ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3;
27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ
ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2,
or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID
NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID
NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID
NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and
210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID
NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2;
262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ
ID NO: 1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions: 4-33
of SEQ ID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID
NO:2, 77-103 of SEQ ID NO: 1 or SEQ ID NO:2, 128-168 of SEQ ID NO:
1 or SEQ ID NO:2, 160-183 of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of
SEQ ID NO: 1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID
NO:2; and wherein the Shiga toxin effector polypeptide is capable
of routing to a cytosol compartment of a cell in which the Shiga
toxin effector polypeptide is present.
123. The polypeptide of claim 119, wherein the Shiga toxin effector
polypeptide is derived from a polypeptide selected from the group
of polypeptides represented by: (i) amino acids 75 to 251 of SEQ ID
NO:1, SEQ ID NO:2, or SEQ ID NO:3; (ii) amino acids 1 to 241 of SEQ
ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; (iii) amino acids 1 to 251
of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and (iv) amino acids 1
to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
124. A method of creating a CD8+ T-cell epitope delivery molecule
capable of intracellular delivery of a T-cell epitope from an early
endosomal compartment to a MHC class I molecule of a cell in which
the delivery molecule is present, the method comprising the step
of: embedding a heterologous, CD8+ T-cell epitope in a proteasome
delivering effector polypeptide capable of intracellular delivery
of a T-cell epitope from an early endosomal compartment to a MHC
class I molecule of a cell in which the delivery molecule is
present; wherein the step of embedding involves replacing an
equivalent number of amino acid residues in a parental polypeptide
with the heterologous, CD8+ T-cell epitope such that the
polypeptide comprising the epitope has the same total number of
amino acids as the parental polypeptide.
125. The method of claim 124, wherein the method comprises
embedding the CD8+ T-cell epitope in an endogenous B-cell epitope,
an endogenous CD4+ T-cell epitope, and/or a catalytic domain of the
proteasome delivering effector polypeptide.
126. The method of claim 124 or claim 125, wherein the CD8+ T-cell
epitope delivery molecule comprises a toxin effector polypeptide
comprising a proteasome delivering effector polypeptide.
127. The method of claim 126, wherein the method comprises
embedding the heterologous, CD8+ T-cell epitope in the toxin
effector polypeptide.
128. The method of claim 127, wherein the embedding step results in
a CD8+ T-cell epitope delivery molecule comprising a toxin effector
polypeptide capable of exhibiting one or more toxin effector
functions in addition to intracellular delivery of a CD8+ T-cell
epitope from an early endosomal compartment to a MHC class I
molecule of a cell in which the toxin effector polypeptide is
present.
129. A method for reducing B-cell immunogenicity of a polypeptide
having a B-cell epitope, the method comprising the step of:
disrupting the B-cell epitope in the polypeptide with one or more
amino acid residue(s) of a T-cell epitope embedded in the
polypeptide; wherein the T-cell epitope is embedded in the
polypeptide by replacing an equivalent number of amino acid
residues in a parental polypeptide with the T-cell epitope such
that the polypeptide comprising the epitope has the same total
number of amino acids as the parental polypeptide.
130. A method for reducing B-cell immunogenicity of a polypeptide
having a B-cell epitope while simultaneously increasing CD8+ T-cell
immunogenicity of the polypeptide, the method comprising the step
of: disrupting a B-cell epitope in the polypeptide with one or more
amino acid residue(s) of a heterologous, CD8+ T-cell epitope
embedded in the polypeptide; wherein the CD8+ T-cell epitope is
embedded in the polypeptide by replacing an equivalent number of
amino acid residues in a parental polypeptide with the
heterologous, CD8+ T-cell epitope such that the polypeptide
comprising the epitope has the same total number of amino acids as
the parental polypeptide.
131. The method of claim 129 or claim 130, wherein the polypeptide
has a CD4+ T-cell epitope, and wherein the B-cell epitope
disrupting step comprises making one or more amino acid
substitutions in the CD4+ T-cell epitope.
132. A method for reducing CD4+ T-cell immunogenicity of a
polypeptide having a CD4+ T-cell epitope, the method comprising the
step of: disrupting a CD4+ T-cell epitope in the polypeptide with
one or more amino acid residue(s) of a CD8+ T-cell epitope embedded
in the polypeptide; wherein the CD8+ T-cell epitope is embedded in
the polypeptide by replacing an equivalent number of amino acid
residues in a parental polypeptide with the CD8+ T-cell epitope
such that the polypeptide comprising the epitope has the same total
number of amino acids as the parental polypeptide.
133. A method for reducing CD4+ T-cell immunogenicity of a
polypeptide having a CD4+ T-cell epitope while simultaneously
increasing CD8+ T-cell immunogenicity of the polypeptide, the
method comprising the step of: disrupting a CD4+ T-cell epitope
with one or more amino acid residue(s) of a heterologous, CD8+
T-cell epitope embedded in the polypeptide; wherein the CD8+ T-cell
epitope is embedded in the polypeptide by replacing an equivalent
number of amino acid residues in a parental polypeptide with the
heterologous, CD8+ T-cell epitope such that the polypeptide
comprising the epitope has the same total number of amino acids as
the parental polypeptide.
134. The method of claim 132 or claim 133, wherein the polypeptide
has a B-cell epitope, and wherein the CD4+ T-cell epitope
disrupting step comprises making one or more amino acid
substitutions in the B-cell epitope.
135. A polypeptide comprising or consisting essentially of the
polypeptide shown in any one of SEQ ID NOs: 11-13, 15-19, 21-43, or
46-48.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to methods of
modifying polypeptides to introduce the ability of the polypeptide
to deliver a heterologous T-cell epitope for MHC class I
presentation by a chordate cell and the polypeptides made using
these methods. More specifically, the invention relates to methods
of modifying polypeptides comprising proteasome delivery effector
functions into heterologous, T-cell epitope delivering polypeptides
that differ in their immunogenic properties from their parent
molecules by the addition of one or more T-cell epitope-peptides
which can be recognized by a MHC class I molecule and be presented
on a cell surface by the MHC class I system of a chordate cell.
Certain methods of the present invention relate to methods of
modifying polypeptides to reduce antigenicity and/or immunogenicity
via the introduction of one or more T-cell epitopes. In another
aspect, the present invention relates to polypeptides created using
methods of the invention and cell-targeted molecules comprising
polypeptides created using methods of the invention. The
cell-targeted molecules of the present invention may be used for
numerous applications such as, e.g., the diagnosis and treatment of
a variety of diseases, disorders, and conditions, such as, e.g.,
cancers, tumors, other growth abnormalities, immune disorders, and
microbial infections.
BACKGROUND
[0002] The immune systems of chordates, such as amphibians, birds,
fish, mammals, reptiles, and sharks, constantly scan both the
extracellular and intracellular environments for exogenous
molecules in an attempt to identify the presence of particularly
threatening foreign molecules, cells, and pathogens. The Major
Histo-Compatibility (MHC) system functions in chordates as part of
the adaptive immune system (Janeway's Immunobiology (Murphy K, ed.,
Garland Science, 8.sup.th ed., 2011)). Within a chordate,
extracellular antigens are presented by the MHC class II system,
whereas intracellular antigens can be presented by the MHC class I
system.
[0003] Generally, the administration of exogenous peptides,
polypeptides, or proteins to a cell results in these molecules not
entering the cell due to the physical barrier of the plasma
membrane. In addition, these molecules are often degraded into
smaller molecules by extracellular enzymatic activities on the
surfaces of cells and/or in the extracellular milieu. Polypeptides
and proteins that are internalized from the extracellular
environment by endocytosis are commonly degraded by lysosomal
proteolysis as part of an endocytotic pathway involving early
endosomes, late endosomes, and lysosomes. Polypeptides and proteins
that are internalized from the extracellular environment by
phagocytosis are commonly degraded by a similar pathway ending with
phagolysosomes
[0004] The MHC class II pathway presents antigenic peptides derived
from molecules in the extracellular space, commonly after
phagocytosis and processing by specialized antigen presenting
cells; these cells can be professional antigen presenting cells or
other antigen presenting cells, such as, e.g., dendritic cells
(DCs), mononuclear phagocytes (MNPCs), certain endothelial cells,
and B-lymphocytes (B-cells). These antigen presenting cells display
certain peptides complexed with MHC class II molecules on their
cell surface for recognition by CD4 positive (CD4+) T-lymphocytes
(T-cells). On the other hand, the MHC class I system functions in
most cells in a chordate to present antigenic peptides from an
intracellular space, commonly the cytosol, for recognition by CD8+
T-cells.
[0005] The MHC class I system plays an essential role in the immune
system by providing antigen presentation of intracellular antigens
(Cellular and Molecular Immunology (Abbas A, ed., Saunders,
8.sup.th ed., 2014)). This process is thought to be an important
part of the adaptive immune system which evolved in chordates
primarily to protect against neoplastic cells and microbial
infections involving intracellular pathogens; however, certain
damaged cells can be removed by this process as well. The
presentation of an antigenic peptide complexed with a MHC class I
molecule sensitizes the presenting cells to targeted killing by
cytotoxic T-cells (CTLs) via lysis, induced apoptosis, and/or
necrosis. The presentation of specific peptide epitopes complexed
with MHC class I molecules plays a major role in stimulating and
maintaining immune responses to cancers, tumors, and intracellular
pathogens.
[0006] The MHC class I system continually functions to process and
display on the cell surface various intracellular epitopes, both
self or non-self (foreign) and both peptide or lipid antigens. The
MHC class I display of foreign antigens from intracellular
pathogens or transformed cells signal to CD8+ effector T-cells to
mount protective T-cell immune responses. In addition, the MHC
class I system continually functions to present self peptide
epitopes in order to establish and maintain immunological
tolerance.
[0007] Peptide epitope presentation by the MHC class I system
involves five main steps: 1) generation of cytoplasmic peptides, 2)
transport of peptides to the lumen of the endoplasmic reticulum
(ER), 3) stable complex formation of MHC class I molecules bound to
certain peptides, 4) display of those stable peptide-MHC class I
molecule complexes (peptide-MHC class I complexes) on the cell
surface, and 5) recognition of certain antigenic, presented
peptide-MHC class I complexes by specific CD8+ T-cells, including
specific CTLs.
[0008] The recognition of a presented antigen-MHC class I complex
by a CD8+ T-cell leads to CD8+ T-cell activation, clonal expansion,
and differentiation into CD8+ effector cells, including CTLs which
target for destruction cells presenting specific epitope-MHC class
I complexes. This leads to the creation of a population of specific
CD8+ effector cells, some of which can travel throughout the body
to seek and destroy cells displaying a specific epitope-MHC class I
complex.
[0009] The MHC class I system is initiated with a cytosolic
peptide. The existence of peptides in the cytosol can occur in
multiple ways. In general, peptides presented by MHC class I
molecules are derived from the proteasomal degradation of
intracellular proteins and polypeptides. The MHC class I pathway
can begin with transporters associated with antigen processing
proteins (TAPs) associated with the ER membrane. TAPs translocate
peptides from the cytosol to the lumen of the ER, where they can
then associate with empty MHC class I molecules. TAPs translocate
peptides which most commonly are of sizes around 8-12 amino acid
residues but also including 6-40 amino acid residues (Koopmann J et
al., Eur J Immunol 26: 1720-8 (1996)).
[0010] The MHC class I pathway can also be initiated in the lumen
of the ER by a pathway involving transport of a protein,
polypeptide, or peptide into the cytosol for processing and then
re-entry back into the ER via TAP-mediated translocation.
[0011] The peptides transported from the cytosol into the lumen of
the ER by TAP are then available to be bound by different MHC class
I molecules. In the lumen of the ER, a multi-component peptide
loading machine, which involves TAPs, helps assemble stable
peptide-MHC class I molecule complexes and further process peptides
in some instances, especially by cleavage into optimal sized
peptides in a process called trimming (see Mayerhofer P, Tampe R, J
Mol Biol pii S0022-2835 (2014)). In the ER, different MHC class I
molecules tightly bind using highly specific immunoglobulin-type,
antigen-binding domains to only those specific peptides for which
the MHC class I molecule has a stronger affinity. Then the
peptide-MHC class I complex is transported via the secretory
pathway to the plasma membrane for presentation to the
extracellular environment and recognition by CD8+ T-cells.
[0012] Recognition by a CD8+ T-cell of an epitope-MHC class I
complex initiates protective immune responses which ultimately ends
in the death of the presenting cell due to the cytotoxic activity
of one or more CTLs. CTLs express different T-cell receptors (TCRs)
with differing specificities. The MHC alleles are highly variable,
and the diversity conferred by these polymorphisms can influence
recognition by T-cells in two ways: by affecting the binding of
peptide antigens and by affecting the contact regions between the
MHC molecule and TCRs. In response to antigen-MHC class I molecule
complex recognition by a CTL via its particular cell surface TCR,
the CTL will kill the antigen-MHC class I complex presenting cell
primarily via cytolytic activities mediated by the delivery of
perforin and/or granzyme into the presenting cell. In addition, the
CTL will release immuno-stimulatory cytokines, such as, e.g.,
interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF),
macrophage inflammatory protein-1 beta (MIP-1beta), and
interleukins such as IL-17, IL-4, and IL-22. Furthermore, activated
CTLs can indiscriminately kill proximal to epitope-MHC class I
complex presenting cell which activated them regardless of the
proximal cell's present peptide-MHC class I complex repertoire
(Wiedemann A et al., Proc Natl Acad Sci USA 103: 10985-90 (2006)).
These epitope-MHC class I complex induced immune responses could
conceivably be harnessed by therapeutics to kill certain cell-types
within a patient as well as sensitize the immune system to other
proximal cells.
[0013] The MHC class I presentation pathway could be exploited by
various therapeutics in order to induce desired immune responses;
however, there are several barriers to developing such a
technology, including, e.g., delivery through the cell plasma
membrane; escaping the endocytotic pathway and destruction in the
lysosome; and generally avoiding the sequestration, modification,
and/or destruction of foreign polypeptides by the targeted cell
(Sahay G et al., J Control Release 145: 182-195 (2010); Fuchs H et
al., Antibodies 2: 209-35 (2013)).
[0014] In addition, the effectiveness of polypeptide-comprising
therapeutics, e.g. polypeptide based biologics and
biopharmaceuticals, is often curtailed by undesirable immune
responses generated in recipients in response to the therapeutics.
Virtually all polypeptide-based therapeutics induce some level of
immune response after administration to a mammalian subject.
Different levels of immune responses include the production of
low-level, low-affinity and transient immunoglobulin-M antibodies
to high-level, high-affinity immunoglobulin-G antibodies. The
immunogenicity of a therapeutic might cause unwanted immune
responses in recipients which reduce therapeutic efficacy,
adversely alter pharmacokinetics, and/or result in hypersensitivity
reactions, anaphylaxis, anaphylactoid reactions, or infusion
reactions among other consequences (see Buttel I et al.,
Biologicals 39: 100-9 (2011)).
[0015] For example, a polypeptide-based therapeutic can cause a
recipient to create antibodies against antigenic sites in the
therapeutic (sometimes called neutralizing antibodies or anti-drug
antibodies). Immune responses generating antibodies recognizing a
therapeutic can result immunological resistance to the effect(s) of
the therapeutic. In addition, cross-reactions between
anti-therapeutic antibodies with endogenous factors can result in
undesirable clinical outcomes.
[0016] Polypeptide-based therapeutics with polypeptide sequences
derived from species distantly related to the recipient, such as
when the recipient is a mammal and the polypeptide sequences are
derived from a plant or microorganism, tend to be aggressively
targeted by the recipient's immune system (see, Sauerborn M et al.,
Trends Pharmacol Sci 31: 53-9 (2010), for review). Vertebrate
immune systems have adapted to recognize foreign polypeptide
sequences with both innate and adaptive immune systems. Thud, the
administration of a polypeptide to a vertebrate from the same
species of vertebrate can be recognized as non-self and elicit an
immune response, such as, e.g., administering to a human a
polypeptide comprising a recombinant junction of two heterologous
human polypeptide sequences.
[0017] Therefore, when designing polypeptide-containing
therapeutics it is often desirable to attempt to minimize the
immunogenicity of the therapeutic to prevent and/or reduce the
occurrence of undesired immune responses in subjects undergoing
therapeutic treatment. In particular, polypeptide regions in
therapeutics likely to produce B-cell and/or T-cell antigenicity
and/or immunogenicity are targeted for removal, suppression, and
minimization.
[0018] Both B-cell and T-cell epitopes can be predicted in a given
polypeptide sequence in silico using software (see, Bryson C et
al., BioDrugs 24: 1-8 (2010), for review). For example, software
called EpiMatrix (EpiVax, Inc., Providence, R.I., U.S.) was
successfully used to predict T-cell immunogenicity in recombinant
proteins (De Groot A et al., Dev Biol (Basel) 122: 171-94 (2005);
Koren E et al., Clin Immunol 124: 26-32 (2007)).
[0019] Many approaches, such as the elimination of antigenic and/or
immunogenic epitopes by truncation or mutation, have been described
for reducing the immunogenicity of polypeptide-containing
therapeutics (Tangri S et al., J Immunol 174: 3187-96 (2005); Mazor
R et al., Proc Natl Acad Sci USA 109: E3597-603 (2012); Yumura K et
al., Protein Sci 22: 213-21 (2012)). Foreign polypeptides can be
recognized with exquisite specificity by the adaptive immune system
via immune epitopes often present at a small number of discrete
sites on the surface of the polypeptide. However, antibody-binding
affinity can be dominated by interactions with a small number of
specific amino acids within an epitope. Thus, modifications of the
crucial amino acids in a polypeptide which disrupt an immunogenic
epitope can reduce immunogenicity (Laroche Y et al., Blood 96:
1425-32 (2000)). Modifications which disrupt epitope recognition
include amino acid deletions, substitutions, and epitope masking
with non-immunogenic conjugates.
[0020] For the development of polypeptide-based therapeutics, it is
desirable to avoid inducing B-cell mediated immune responses and
the production of neutralizing antibodies in patients because it
reduces the effectiveness of the therapy, changes the dose-effect
profile, and limits the number of doses a patient can receive (see
Lui W et al., Proc Natl Acad Sci USA 109: 11782-7 (2012)).
[0021] Thus, it would be desirable to have methods of creating
novel T-cell epitope delivering polypeptides which can deliver one
or more T-cell epitopes to the MHC class I presentation pathway of
a cell. It would also be desirable to have polypeptides which under
physiological conditions can deliver a T-cell epitope to the
interior of a target cell to initiate desirable T-cell mediated
immune responses but do not induce undesirable immune responses
while in extracellular spaces, such as, e.g., the creation of
inhibitory antibodies. Thus, it would be desirable to have T-cell
epitope delivering polypeptides in which one or more CD8+ T-cell
epitopes are added and one or more B-cell and/or CD4+ T-cell
epitopes are abolished.
[0022] It would also be desirable to have cell-targeted, CD8+
T-cell epitope delivering molecules for the targeted delivery of
cytotoxicity to specific cell types, e.g., infected or malignant
cells. In addition, it would be desirable to have cell-targeted,
CD8+ T-cell epitope delivering molecules which exhibit reduced
B-cell immunogenicity. Once the T-cell immunogenic peptide(s)
delivered by the cell-targeted molecule are presented to the
surface of a target cell, the T-cell epitope can signal for the
destruction of the presenting cell by activating the recipient's
own immune system to recruit CD8+ T-cells. In addition, CD8+
T-cells activated by the target cell's displayed T-cell epitope-MHC
class I complex can stimulate a wider immune response and alter the
micro-environment (e.g. by release cytokines in a tumor or infected
tissue locus), such that other immune cells (e.g. effector T-cells)
may be recruited to the local area.
[0023] In addition, it would be desirable to have methods of
creating novel T-cell epitope delivering polypeptides which are
derived from toxins yet preserving certain biological effector
functions of the parental toxin polypeptide, such as promoting
cellular internalization, directing subcellular routing, and/or
toxin enzymatic activity. In addition, it is desirable to have
methods of engineering toxin-derived polypeptides by replacing a
B-cell epitope with a T-cell epitope as a means to both reduce the
likelihood of the polypeptide producing an undesirable immune
response and to increase the likelihood of inducing a desirable
T-cell response directed to those targeted cells that internalize
the toxin polypeptide comprising molecule.
SUMMARY OF THE INVENTION
[0024] The present invention provides various embodiments of T-cell
epitope delivering polypeptides (referred to herein as "CD8+ T-cell
hyper-immunized") which as components of certain cell-targeted
molecules have the ability to deliver a T-cell epitope for
presentation by a nucleated, target cell within a chordate. The
present invention also provides various embodiments of
de-immunized, CD8+ T-cell hyper-immunized polypeptides which have
reduced antigenic and/or immunogenic potential in mammals regarding
a B-cell and/or CD4+ T-cell epitope (referred to herein as "B-cell
and/or CD4+ T-cell de-immunized"). The present invention also
provides various embodiments of cell-targeted, CD8+ T-cell epitope
delivering molecules for the targeted delivery of cytotoxicity to
specific cell types, e.g., infected or malignant cells within a
chordate.
[0025] In addition, the present invention provides embodiments of
methods of generating novel polypeptides capable of delivering one
or more heterologous T-cell epitopes to the MHC class I
presentation pathway of a cell. The present invention also provides
various embodiments of methods of generating variants of
polypeptides by simultaneously reducing the probability of B-cell
and/or CD4+ T-cell immunogenicity while increasing the probability
of CD8+ T-cell immunogenicity. The present invention also provides
certain embodiments of the methods of generating novel polypeptides
capable of delivering one or more heterologous T-cell epitopes to
the MHC class I presentation pathway of a cell, wherein the
starting polypeptide comprises a toxin effector region and certain
polypeptides produced by using the methods of the invention result
in polypeptides which retain toxin effector functions, such as,
e.g., enzymatic activity and cytotoxicity.
[0026] The polypeptides of the present invention may be either CD8+
T-cell hyper-immunized or de-immunized or both. The de-immunized
polypeptides of the present invention may be either B-cell epitope
de-immunized or T-cell de-immunized or both. The T-cell
de-immunized polypeptides of the present invention may be either
CD4+ T-cell de-immunized or CD8+ T-cell de-immunized or both.
Certain embodiments of the polypeptides of the present invention
comprise one or more heterologous T-cell epitopes. In certain
further embodiments of the polypeptides of the present invention,
the one or more heterologous T-cell epitopes are CD8+ T-cell
epitopes.
[0027] In certain embodiments, a polypeptide of the present
invention comprises an embedded or inserted heterologous T-cell
epitope, wherein the polypeptide is capable of intracellular
delivery of the T-cell epitope from an early endosomal compartment
to a proteasome of a cell in which the polypeptide is present. In
certain further embodiments, the polypeptide of the present
invention further comprises a toxin-derived polypeptide capable of
routing to a subcellular compartment of a cell in which the
toxin-derived polypeptide is present selected from the group
consisting of: cytosol, endoplasmic reticulum, and lysosome. In
certain further embodiments, the polypeptide of the present
invention comprises a heterologous T-cell epitope is embedded or
inserted in a toxin-derived polypeptide.
[0028] In certain embodiments, a polypeptide of the present
invention comprises a toxin-derived polypeptide comprising a toxin
effector polypeptide capable of exhibiting one or more toxin
effector functions. In certain further embodiments, the toxin
effector polypeptide is derived from a toxin selected from the
group consisting of: ABx toxin, ribosome inactivating protein
toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix
toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin,
mitogillin, pertussis toxin, pokeweed antiviral protein,
pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,
sarcin, Shiga toxin, and subtilase cytotoxin.
[0029] In certain embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from
amino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3,
or amino acids 2 to 389 of SEQ ID NO:45. In certain further
embodiments, the polypeptide of the present invention comprises the
Shiga toxin effector polypeptide derived from amino acids 1 to 241
of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further
embodiments, the Shiga toxin effector polypeptide is derived from
amino acids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
In certain further embodiments, the Shiga toxin effector
polypeptide is derived from amino acids 1 to 261 of SEQ ID NO:1,
SEQ ID NO:2, or SEQ ID NO:3.
[0030] In certain embodiments, a polypeptide of the present
invention comprises an embedded or inserted heterologous CD8+
T-cell epitope, wherein the polypeptide is capable of intracellular
delivery of the T-cell epitope to a MHC class I molecule from an
early endosomal compartment of a cell in which the polypeptide is
present. In certain further embodiments, the polypeptide further
comprises a toxin-derived polypeptide capable of routing to a
subcellular compartment of a cell in which the polypeptide is
present selected from the group consisting of: cytosol, endoplasmic
reticulum, and lysosome. In certain further embodiments, the
polypeptide of the present invention comprises the heterologous
CD8+ T-cell epitope in the toxin-derived polypeptide. In certain
further embodiments, the polypeptide of the present invention
comprises the toxin-derived polypeptide comprising a toxin effector
polypeptide capable of exhibiting one or more toxin effector
functions. In certain further embodiments, the polypeptide of the
present invention comprises the toxin effector polypeptide derived
from a toxin selected from the group consisting of: ABx toxin,
ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl,
bouganin, bryodin, cholix toxin, claudin, diphtheria toxin,
gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin,
pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from
amino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3,
or amino acids 2 to 389 of SEQ ID NO:45. In certain further
embodiments, the polypeptide of the present invention comprises the
Shiga toxin effector polypeptide derived from amino acids 1 to 241
of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further
embodiments, the Shiga toxin effector polypeptide is derived from
amino acids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
In certain further embodiments, the Shiga toxin effector
polypeptide is derived from amino acids 1 to 261 of SEQ ID NO:1,
SEQ ID NO:2, or SEQ ID NO:3.
[0031] In certain embodiments, a polypeptide of the present
invention comprises a heterologous CD8+ T-cell epitope, wherein the
polypeptide is capable of intracellular delivery of the T-cell
epitope for presentation by a MHC class I molecule on the surface
of a cell in which the polypeptide is present. In certain further
embodiments, the polypeptide of the present invention comprises a
toxin-derived polypeptide capable of routing to a subcellular
compartment of a cell in which the toxin-derived polypeptide is
present selected from the group consisting of: cytosol, endoplasmic
reticulum, and lysosome. In certain further embodiments, the
polypeptide of the present invention comprises the heterologous
CD8+ T-cell epitope in the toxin-derived polypeptide. In certain
further embodiments, the polypeptide of the present invention
comprises the toxin-derived polypeptide comprising a toxin effector
polypeptide capable of exhibiting one or more toxin effector
functions. In certain further embodiments, the polypeptide of the
present invention comprises the toxin effector polypeptide derived
from a toxin selected from the group consisting of: ABx toxin,
ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl,
bouganin, bryodin, cholix toxin, claudin, diphtheria toxin,
gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin,
pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from
amino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3,
or amino acids 2 to 389 of SEQ ID NO:45. In certain further
embodiments, the polypeptide of the present invention comprises the
Shiga toxin effector polypeptide derived from amino acids 1 to 241
of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further
embodiments, the Shiga toxin effector polypeptide is derived from
amino acids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
In certain further embodiments, the Shiga toxin effector
polypeptide is derived from amino acids 1 to 261 of SEQ ID NO:1,
SEQ ID NO:2, or SEQ ID NO:3.
[0032] In certain embodiments, a polypeptide of the present
invention comprises a proteasome delivering effector polypeptide
associated with a heterologous CD8+ T-cell epitope, and capable of
intracellular delivery of the T-cell epitope for presentation by a
MHC class I molecule on the surface of a cell in which the
polypeptide is present. In certain further embodiments, the
polypeptide of the present invention comprises a Shiga toxin
effector polypeptide, wherein the heterologous CD8+ T-cell epitope
is not fused directly to the amino-terminus of the Shiga toxin
effector polypeptide. In certain further embodiments, the
polypeptide of the present invention further comprises a second
T-cell epitope embedded or inserted into a B-cell epitope. In
certain further embodiments, the polypeptide of the present
invention further comprises a toxin-derived polypeptide. In certain
further embodiments, the polypeptide of the present invention
further comprises the toxin-derived polypeptide comprising a toxin
effector polypeptide comprising the proteasome delivering effector
polypeptide and the second T-cell epitope. In certain further
embodiments, a polypeptide of the present invention comprises the
toxin-derived polypeptide comprising a toxin effector polypeptide
capable of exhibiting one or more toxin effector functions. In
certain further embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from a
toxin selected from the group consisting of: ABx toxin, ribosome
inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,
bryodin, cholix toxin, claudin, diphtheria toxin, gelonin,
heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed
antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain embodiments, the polypeptide of 15 the
present invention comprises the toxin effector polypeptide derived
from amino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID
NO:3, or amino acids 2 to 389 of SEQ ID NO:45. In certain further
embodiments, the polypeptide of the present invention comprises the
Shiga toxin effector polypeptide derived from amino acids 1 to 241
of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In certain further
embodiments, the Shiga toxin effector polypeptide is derived from
amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
In certain further embodiments, the Shiga toxin effector
polypeptide is derived from amino acids 1 to 261 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3.
[0033] In certain embodiments of the methods of the present
invention is a method of increasing CD8+ T-cell immunogenicity of a
polypeptide capable of intracellular routing to a subcellular
compartment of a cell in which the polypeptide is present selected
from the group consisting of: cytosol, endoplasmic reticulum, and
lysosome; the method comprising the step of: embedding or inserting
a heterologous CD8+ T-cell epitope in the polypeptide. In certain
further embodiments, the method comprises the embedding or
inserting step wherein the embedding or inserting in an endogenous
B-cell epitope, an endogenous CD4+ T-cell epitope, and/or a
catalytic domain of the polypeptide. In certain further embodiments
of the method, the polypeptide of the method is derived from a
toxin. In certain further embodiments of the method, the
polypeptide comprises a toxin effector polypeptide capable of
intracellular delivery of a T-cell epitope from an early endosomal
compartment to a proteasome of a cell in which the toxin effector
polypeptide is present, and the method comprises embedding or
inserting the heterologous T-cell epitope in the toxin effector
polypeptide. In certain further embodiments of the method, the
embedding or inserting step results in a toxin effector polypeptide
capable of exhibiting one or more toxin effector functions in
addition to intracellular delivery of a T-cell epitope from an
early endosomal compartment to a MHC class I molecule of a cell in
which the toxin effector polypeptide is present.
[0034] In certain embodiments of the methods of the present
invention is a method of increasing CD8+ T-cell immunogenicity of a
polypeptide capable of intracellular delivery of a T-cell epitope
from an early endosomal compartment to a proteasome of a cell in
which the polypeptide is present, the method comprising the step
of: embedding or inserting a heterologous CD8+ T-cell epitope in
the polypeptide. In certain further embodiments of the method, the
polypeptide of the method is derived from a toxin. In certain
further embodiments of the method, the polypeptide comprises a
toxin effector polypeptide capable of intracellular delivery of a
T-cell epitope from an early endosomal compartment to a proteasome
of a cell in which the toxin effector polypeptide is present, and
the method comprises embedding or inserting the heterologous T-cell
epitope in the toxin effector polypeptide. In certain further
embodiments of the method, the embedding or inserting step results
in a toxin effector polypeptide capable of exhibiting one or more
toxin effector functions in addition to intracellular delivery of a
T-cell epitope from an early endosomal compartment to a MHC class I
molecule of a cell in which the toxin effector polypeptide is
present.
[0035] In certain embodiments of the methods of the present
invention is a method of increasing CD8+ T-cell immunogenicity of a
polypeptide capable of intracellular delivery of a T-cell epitope
from an early endosomal compartment to a MHC class I molecule of a
cell in which the polypeptide is present, the method comprising the
step of: embedding or inserting a heterologous CD8+ T-cell epitope
in the polypeptide. In certain further embodiments of the method,
the polypeptide of the method is derived from a toxin. In certain
further embodiments of the method, the polypeptide comprises a
toxin effector polypeptide capable of intracellular delivery of a
T-cell epitope from an early endosomal compartment to a proteasome
of a cell in which the toxin effector polypeptide is present, and
the method comprises embedding or inserting the heterologous T-cell
epitope in the toxin effector polypeptide. In certain further
embodiments of the method, the embedding or inserting step results
in a toxin effector polypeptide capable of exhibiting one or more
toxin effector functions in addition to intracellular delivery of a
T-cell epitope from an early endosomal compartment to a MHC class I
molecule of a cell in which the toxin effector polypeptide is
present.
[0036] In certain embodiments of the methods of the present
invention is a method of creating a T-cell epitope delivery
molecule capable of intracellular delivery of a T-cell epitope from
an early endosomal compartment to the cytosol, endoplasmic
reticulum, and/or lysosome of a cell in which the molecule is
present, the method comprising the step of: associating a
heterologous T-cell epitope with a polypeptide capable of routing
to a subcellular compartment of a cell in which the polypeptide is
present selected from the group consisting of: cytosol, endoplasmic
reticulum, and lysosome. In certain further embodiments of the
method, the associating consists of embedding or inserting the
heterologous T-cell epitope in an endogenous B-cell epitope, an
endogenous CD4+ T-cell epitope, and/or a catalytic domain of the
molecule. In certain further embodiments of the method, the
polypeptide of the method is derived from a toxin. In certain
further embodiments of the method, the polypeptide comprises a
toxin effector polypeptide capable of intracellular delivery of a
T-cell epitope from an early endosomal compartment to the cytosol,
endoplasmic reticulum, and/or lysosome of a cell in which the toxin
effector polypeptide is present, and the method comprises embedding
or inserting the heterologous T-cell epitope in the toxin effector
polypeptide. In certain further embodiments of the method, the
embedding or inserting step results in a toxin effector polypeptide
capable of exhibiting one or more toxin effector functions in
addition to intracellular delivery of a T-cell epitope from an
early endosomal compartment to the cytosol, endoplasmic reticulum,
and/or lysosome of a cell in which the toxin effector polypeptide
is present.
[0037] In certain embodiments of the methods of the present
invention is a method of creating a CD8+ T-cell epitope delivery
molecule capable of intracellular delivery of a T-cell epitope from
an early endosomal compartment to a proteasome of a cell in which
the delivery molecule is present, the method comprising the step
of: embedding or inserting a heterologous CD8+ T-cell epitope in a
proteasome delivering effector polypeptide capable of intracellular
delivery of a T-cell epitope from an early endosomal compartment to
a proteasome of a cell in which the proteasome delivering effector
polypeptide is present. In certain further embodiments of the
method, the associating consists of embedding or inserting the
heterologous T-cell epitope in an endogenous B-cell epitope, an
endogenous CD4+ T-cell epitope, and/or a catalytic domain of the
molecule. In certain further embodiments of the method, the
polypeptide of the method is derived from a toxin. In certain
further embodiments of the method, the polypeptide comprises a
toxin effector polypeptide capable of exhibiting one or more toxin
effector functions in addition to intracellular delivery of a
T-cell epitope from an early endosomal compartment to a proteasome
of a cell in which the toxin effector polypeptide is present.
[0038] In certain embodiments of the methods of the present
invention is a method of creating a CD8+ T-cell epitope delivery
molecule capable of intracellular delivery of a T-cell epitope from
an early endosomal compartment to a MHC class I molecule of a cell
in which the delivery molecule is present, the method comprising
the step of: embedding or inserting a heterologous CD8+ T-cell
epitope in a proteasome delivering effector polypeptide capable of
intracellular delivery of a T-cell epitope from an early endosomal
compartment to a MHC class I molecule of a cell in which the
proteasome delivering effector polypeptide is present. In certain
further embodiments of the method, the associating consists of
embedding or inserting the heterologous T-cell epitope in an
endogenous B-cell epitope, an endogenous CD4+ T-cell epitope,
and/or a catalytic domain of the molecule. In certain further
embodiments of the method, the polypeptide of the method is derived
from a toxin. In certain further embodiments of the method, the
polypeptide comprises a toxin effector polypeptide comprising the
proteasome delivering effector polypeptide, and the method
comprises embedding or inserting the heterologous T-cell epitope in
the toxin effector polypeptide. In certain further embodiments of
the method, the toxin effector polypeptide resulting from the is
capable of exhibiting one or more toxin effector functions in
addition to intracellular delivery of a T-cell epitope from an
early endosomal compartment to a MHC class I molecule of a cell in
which the toxin effector polypeptide is present.
[0039] In certain embodiments of the methods of the present
invention is a method of creating a CD8+ T-cell epitope delivery
molecule capable when present in a cell of delivering a T-cell
epitope for presentation by a MHC class I molecule, the method
comprising the step of: embedding or inserting a heterologous CD8+
T-cell epitope in a proteasome delivering effector polypeptide
capable of intracellular delivery of a T-cell epitope from an early
endosomal compartment to a proteasome of a cell in which the
proteasome delivering effector polypeptide is present. In certain
further embodiments of the method, the associating consists of
embedding or inserting the heterologous T-cell epitope in an
endogenous B-cell epitope, an endogenous CD4+ T-cell epitope,
and/or a catalytic domain of the molecule. In certain further
embodiments of the method, the polypeptide of the method is derived
from a toxin. In certain further embodiments of the method, the
polypeptide comprises a toxin effector polypeptide comprising the
proteasome delivering effector polypeptide, and the method
comprises embedding or inserting the heterologous T-cell epitope in
the toxin effector polypeptide. In certain further embodiments of
the method, the toxin effector polypeptide resulting from the is
capable of exhibiting one or more toxin effector functions in
addition to intracellular delivery of a T-cell epitope from an
early endosomal compartment to a MHC class I molecule of a cell in
which the toxin effector polypeptide is present.
[0040] In certain embodiments of the methods of the present
invention is a method of creating a CD8+ T-cell epitope delivery
molecule capable when present in a cell of delivering a T-cell
epitope for presentation by a MHC class I molecule, the method
comprising the step of: embedding or inserting a heterologous CD8+
T-cell epitope in a proteasome delivering effector polypeptide
capable of intracellular delivery of a T-cell epitope from an early
endosomal compartment to a MHC class I molecule of a cell in which
the proteasome delivering effector polypeptide is present. In
certain further embodiments of the method, the associating consists
of embedding or inserting the heterologous T-cell epitope in an
endogenous B-cell epitope, an endogenous CD4+ T-cell epitope,
and/or a catalytic domain of the molecule. In certain further
embodiments of the method, the polypeptide of the method is derived
from a toxin. In certain further embodiments of the method, the
polypeptide comprises a toxin effector polypeptide comprising the
proteasome delivering effector polypeptide, and the method
comprises embedding or inserting the heterologous T-cell epitope in
the toxin effector polypeptide. In certain further embodiments of
the method, the toxin effector polypeptide resulting from the is
capable of exhibiting one or more toxin effector functions in
addition to intracellular delivery of a T-cell epitope from an
early endosomal compartment to a MHC class I molecule of a cell in
which the toxin effector polypeptide is present.
[0041] In certain embodiments, a de-immunized polypeptide of the
present invention comprises a heterologous T-cell epitope
disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope.
In certain further embodiments, the polypeptide of the present
invention comprises a toxin-derived polypeptide. In certain further
embodiments, the heterologous CD8+ T-cell epitope is in the
toxin-derived polypeptide. In certain further embodiments, the
toxin-derived polypeptide of the present invention comprises a
toxin effector polypeptide. In certain further embodiments, the
heterologous CD8+ T-cell epitope in the toxin effector polypeptide.
In certain further embodiments, the toxin effector polypeptide is
capable of exhibiting one or more toxin effector functions. In
certain further embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from a
toxin selected from the group consisting of: ABx toxin, ribosome
inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,
bryodin, cholix toxin, claudin, diphtheria toxin, gelonin,
heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed
antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain further embodiments, the toxin effector
polypeptide is a diphtheria toxin effector polypeptide comprising
an amino acid sequence derived from the A and B Subunits of at
least one member of the diphtheria toxin family, wherein the
diphtheria toxin effector polypeptide comprises a disruption of at
least one B-cell epitope and/or CD4+ T-cell epitope region of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID
NO:39, 71-77 of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of
SEQ ID NO:39, 165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39;
and wherein the diphtheria toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the diphtheria
toxin effector polypeptide is present. In certain further
embodiments, the polypeptide of the present invention comprises the
diphtheria toxin effector polypeptide derived from amino acids 2 to
389 of SEQ ID NO:45. In certain further embodiment, the toxin
effector polypeptide is a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of at
least one member of the Shiga toxin family, wherein the Shiga toxin
effector polypeptide comprises a disruption of at least one B-cell
epitope and/or CD4+ T-cell epitope region of the Shiga toxin A
Subunit amino acid sequence selected from the group of natively
positioned amino acids consisting of: the B-cell epitope regions
1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of
SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID
NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or
SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ
ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID
NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; and
210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID
NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2;
262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ
ID NO:1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of
SEQ ID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2,
77-103 of SEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ
ID NO:2, 160-183 of SEQ ID NO: 1 or SEQ ID NO:2, 236-258 of SEQ ID
NO: 1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2;
and wherein the Shiga toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the Shiga toxin
effector polypeptide is present. In certain embodiments, the
polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 75 to 251 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 1 to 241 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the Shiga toxin effector polypeptide is derived from amino acids 1
to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain
further embodiments, the Shiga toxin effector polypeptide is
derived from amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO:2, or
SEQ ID NO:3.
[0042] In certain embodiments, a polypeptide of the present
invention comprises a heterologous CD8+ T-cell epitope disrupting
an endogenous B-cell epitope and/or an endogenous CD4+ T-cell
epitope, wherein the polypeptide is capable of intracellular
delivery of the CD8+ T-cell epitope from an early endosomal
compartment to a proteasome of a cell in which the polypeptide is
present. In certain further embodiments, the polypeptide of the
present invention comprises a toxin-derived polypeptide. In certain
further embodiments, the heterologous CD8+ T-cell epitope is in the
toxin-derived polypeptide. In certain further embodiments, the
toxin-derived polypeptide of the present invention comprises a
toxin effector polypeptide. In certain further embodiments, the
heterologous CD8+ T-cell epitope in the toxin effector polypeptide.
In certain further embodiments, the toxin effector polypeptide is
capable of exhibiting one or more toxin effector functions. In
certain further embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from a
toxin selected from the group consisting of: ABx toxin, ribosome
inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,
bryodin, cholix toxin, claudin, diphtheria toxin, gelonin,
heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed
antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain further embodiments, the toxin effector
polypeptide is a diphtheria toxin effector polypeptide comprising
an amino acid sequence derived from the A and B Subunits of at
least one member of the diphtheria toxin family, wherein the
diphtheria toxin effector polypeptide comprises a disruption of at
least one B-cell epitope and/or CD4+ T-cell epitope region of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID
NO:39, 71-77 of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of
SEQ ID NO:39, 165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39;
and wherein the diphtheria toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the diphtheria
toxin effector polypeptide is present. In certain further
embodiments, the polypeptide of the present invention comprises the
diphtheria toxin effector polypeptide derived from amino acids 2 to
389 of SEQ ID NO:45. In certain further embodiment, the toxin
effector polypeptide is a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of at
least one member of the Shiga toxin family, wherein the Shiga toxin
effector polypeptide comprises a disruption of at least one B-cell
epitope and/or CD4+ T-cell epitope region of the Shiga toxin A
Subunit amino acid sequence selected from the group of natively
positioned amino acids consisting of: the B-cell epitope regions
1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of
SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID
NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2,
or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of
SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ
ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and
210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID
NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278
of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1
or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQ ID
NO:1 or SEQ ID NO:2, 34-78 of SEQ ID NO:1 or SEQ ID NO:2, 77-103 of
SEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2,
160-183 of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID NO:1 or
SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and
wherein the Shiga toxin effector polypeptide is capable of routing
to a cytosol compartment of a cell in which the Shiga toxin
effector polypeptide is present. In certain embodiments, the
polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 75 to 251 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 1 to 241 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the Shiga toxin effector polypeptide is derived from amino acids 1
to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain
further embodiments, the Shiga toxin effector polypeptide is
derived from amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or
SEQ ID NO:3.
[0043] In certain embodiments, a de-immunized polypeptide of the
present invention comprises a heterologous CD8+ T-cell epitope
disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope,
wherein the polypeptide is capable of intracellular delivery of the
CD8+ T-cell epitope to a MHC class I molecule from an early
endosomal compartment of a cell in which the polypeptide is
present. In certain further embodiments, the polypeptide of the
present invention comprises a toxin-derived polypeptide. In certain
further embodiments, the heterologous CD8+ T-cell epitope is in the
toxin-derived polypeptide. In certain further embodiments, the
toxin-derived polypeptide of the present invention comprises a
toxin effector polypeptide. In certain further embodiments, the
heterologous CD8+ T-cell epitope in the toxin effector polypeptide.
In certain further embodiments, the toxin effector polypeptide is
capable of exhibiting one or more toxin effector functions. In
certain further embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from a
toxin selected from the group consisting of: ABx toxin, ribosome
inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,
bryodin, cholix toxin, claudin, diphtheria toxin, gelonin,
heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed
antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain further embodiments, the toxin effector
polypeptide is a diphtheria toxin effector polypeptide comprising
an amino acid sequence derived from the A and B Subunits of at
least one member of the diphtheria toxin family, wherein the
diphtheria toxin effector polypeptide comprises a disruption of at
least one B-cell epitope and/or CD4+ T-cell epitope region of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID
NO:39, 71-77 of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of
SEQ ID NO:39, 165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39;
and wherein the diphtheria toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the diphtheria
toxin effector polypeptide is present. In certain further
embodiments, the polypeptide of the present invention comprises the
diphtheria toxin effector polypeptide derived from amino acids 2 to
389 of SEQ ID NO:45. In certain further embodiment, the toxin
effector polypeptide is a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of at
least one member of the Shiga toxin family, wherein the Shiga toxin
effector polypeptide comprises a disruption of at least one B-cell
epitope and/or CD4+ T-cell epitope region of the Shiga toxin A
Subunit amino acid sequence selected from the group of natively
positioned amino acids consisting of: the B-cell epitope regions
1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of
SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID
NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or
SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ
ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID
NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; and
210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID
NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2;
262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ
ID NO: 1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33
of SEQ ID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID
NO:2, 77-103 of SEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1
or SEQ ID NO:2, 160-183 of SEQ ID NO: 1 or SEQ ID NO:2, 236-258 of
SEQ ID NO: 1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID
NO:2; and wherein the Shiga toxin effector polypeptide is capable
of routing to a cytosol compartment of a cell in which the Shiga
toxin effector polypeptide is present. In certain embodiments, the
polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 75 to 251 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 1 to 241 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the Shiga toxin effector polypeptide is derived from amino acids 1
to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain
further embodiments, the Shiga toxin effector polypeptide is
derived from amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO:2, or
SEQ ID NO:3.
[0044] In certain embodiments, a de-immunized polypeptide of the
present invention comprises a heterologous CD8+ T-cell epitope
disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope,
wherein the polypeptide is capable of intracellular delivery of the
CD 8+ T-cell epitope for presentation by a MHC class I molecule on
the surface of a cell in which the polypeptide is present. In
certain further embodiments, the polypeptide of the present
invention comprises a toxin-derived polypeptide. In certain further
embodiments, the heterologous CD8+ T-cell epitope is in the
toxin-derived polypeptide. In certain further embodiments, the
toxin-derived polypeptide of the present invention comprises a
toxin effector polypeptide. In certain further embodiments, the
heterologous CD8+ T-cell epitope in the toxin effector polypeptide.
In certain further embodiments, the toxin effector polypeptide is
capable of exhibiting one or more toxin effector functions. In
certain further embodiments, the polypeptide of the present
invention comprises the toxin effector polypeptide derived from a
toxin selected from the group consisting of: ABx toxin, ribosome
inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,
bryodin, cholix toxin, claudin, diphtheria toxin, gelonin,
heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed
antiviral protein, pulchellin, Pseudomonas exotoxin A,
restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain further embodiments, the toxin effector
polypeptide is a diphtheria toxin effector polypeptide comprising
an amino acid sequence derived from the A and B Subunits of at
least one member of the diphtheria toxin family, wherein the
diphtheria toxin effector polypeptide comprises a disruption of at
least one B-cell epitope and/or CD4+ T-cell epitope region of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID
NO:39, 71-77 of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of
SEQ ID NO:39, 165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39;
and wherein the diphtheria toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the diphtheria
toxin effector polypeptide is present. In certain further
embodiments, the polypeptide of the present invention comprises the
diphtheria toxin effector polypeptide derived from amino acids 2 to
389 of SEQ ID NO:45. In certain further embodiment, the toxin
effector polypeptide is a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of at
least one member of the Shiga toxin family, wherein the Shiga toxin
effector polypeptide comprises a disruption of at least one B-cell
epitope and/or CD4+ T-cell epitope region of the Shiga toxin A
Subunit amino acid sequence selected from the group of natively
positioned amino acids consisting of: the B-cell epitope regions
1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of
SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID
NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2,
or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of
SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ
ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2;
and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ
ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2;
262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ
ID NO:1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of
SEQ ID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO:1 or SEQ ID NO:2,
77-103 of SEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ
ID NO:2, 160-183 of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID
NO:1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2;
and wherein the Shiga toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the Shiga toxin
effector polypeptide is present. In certain embodiments, the
polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 75 to 251 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 1 to 241 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the Shiga toxin effector polypeptide is derived from amino acids 1
to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain
further embodiments, the Shiga toxin effector polypeptide is
derived from amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or
SEQ ID NO:3.
[0045] In certain embodiments, a de-immunized polypeptide of the
present invention comprises a proteasome delivering effector
polypeptide comprising a first heterologous T-cell epitope
disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope,
wherein the proteasome delivering effector polypeptide is linked to
a second CD8+ T-cell epitope; and the polypeptide is capable of
intracellular delivery of the second CD8+ T-cell epitope for
presentation by a MHC class I molecule on the surface of a cell in
which the polypeptide is present. In certain further embodiments,
the polypeptide of the present invention comprises a toxin-derived
polypeptide. In certain further embodiments, the heterologous CD8+
T-cell epitope is in the toxin-derived polypeptide. In certain
further embodiments, the toxin-derived polypeptide of the present
invention comprises a toxin effector polypeptide. In certain
further embodiments, the heterologous CD8+ T-cell epitope in the
toxin effector polypeptide. In certain further embodiments, the
toxin effector polypeptide is capable of exhibiting one or more
toxin effector functions. In certain further embodiments, the
polypeptide of the present invention comprises the toxin effector
polypeptide derived from a toxin selected from the group consisting
of: ABx toxin, ribosome inactivating protein toxin, abrin, anthrax
toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria
toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis
toxin, pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin
A, restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase
cytotoxin. In certain further embodiments, the toxin effector
polypeptide is a diphtheria toxin effector polypeptide comprising
an amino acid sequence derived from the A and B Subunits of at
least one member of the diphtheria toxin family, wherein the
diphtheria toxin effector polypeptide comprises a disruption of at
least one B-cell epitope and/or CD4+ T-cell epitope region of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID
NO:39, 71-77 of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of
SEQ ID NO:39, 165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39;
and wherein the diphtheria toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the diphtheria
toxin effector polypeptide is present. In certain further
embodiments, the polypeptide of the present invention comprises the
diphtheria toxin effector polypeptide derived from amino acids 2 to
389 of SEQ ID NO:45. In certain further embodiment, the toxin
effector polypeptide is a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of at
least one member of the Shiga toxin family, wherein the Shiga toxin
effector polypeptide comprises a disruption of at least one B-cell
epitope and/or CD4+ T-cell epitope region of the Shiga toxin A
Subunit amino acid sequence selected from the group of natively
positioned amino acids consisting of: the B-cell epitope regions
1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of
SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID
NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1,
SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2,
or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of
SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ
ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2;
and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ
ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2;
262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ
ID NO:1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of
SEQ ID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO:1 or SEQ ID NO:2,
77-103 of SEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ
ID NO:2, 160-183 of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID
NO:1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2;
and wherein the Shiga toxin effector polypeptide is capable of
routing to a cytosol compartment of a cell in which the Shiga toxin
effector polypeptide is present. In certain embodiments, the
polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 75 to 251 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the polypeptide of the present invention comprises the Shiga toxin
effector polypeptide derived from amino acids 1 to 241 of SEQ ID
NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,
the Shiga toxin effector polypeptide is derived from amino acids 1
to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain
further embodiments, the Shiga toxin effector polypeptide is
derived from amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or
SEQ ID NO:3.
[0046] In certain embodiments of the methods of the present
invention is a method for reducing B-cell immunogenicity in a
polypeptide, the method comprising the step of: disrupting a B-cell
epitope with one or more amino acid residue(s) of a T-cell epitope
added to the polypeptide. In certain further embodiments, the
disrupting step further comprises the step or steps of making one
or more amino acid substitutions in the B-cell epitope. In certain
further embodiments, the disrupting step further comprises the step
or steps of making one or more amino acid insertions in the B-cell
epitope.
[0047] In certain embodiments of the methods of the present
invention is a method for reducing B-cell immunogenicity in a
polypeptide, the method comprising the steps of: identifying a
B-cell epitope in a polypeptide; and disrupting the identified
B-cell epitope with one or more amino acid residue(s) in a T-cell
epitope added to polypeptide. In certain further embodiments, the
disrupting step further comprises the step or steps of making one
or more amino acid substitutions in the B-cell epitope. In certain
further embodiments, the disrupting step further comprises the step
or steps of making one or more amino acid insertions in the B-cell
epitope.
[0048] In certain embodiments of the methods of the present
invention is a method for reducing B-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity of the polypeptide, the method comprising the step
of: disrupting a B-cell epitope with one or more amino acid
residue(s) in a heterologous CD8+ T-cell epitope added to the
polypeptide. In certain further embodiments, the disrupting step
further comprises the step or steps of making one or more amino
acid substitutions in the B-cell epitope. In certain further
embodiments, the disrupting step further comprises the step or
steps of making one or more amino acid insertions in the B-cell
epitope.
[0049] In certain embodiments of the methods of the present
invention is a method for reducing B-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity of the polypeptide, the method comprising the steps
of: identifying a CD4+ T-cell epitope in a polypeptide; and
disrupting the identified CD4+ T-cell epitope with one or more
amino acid residue(s) in a CD8+ T-cell epitope added to the
polypeptide. In certain further embodiments, the disrupting step
further comprises the step or steps of making one or more amino
acid substitutions in the B-cell epitope. In certain further
embodiments, the disrupting step further comprises the step or
steps of making one or more amino acid insertions in the B-cell
epitope.
[0050] In certain embodiments of the methods of the present
invention is method for reducing CD4+ T-cell immunogenicity in a
polypeptide, the method comprising the step of: disrupting a CD4+
T-cell epitope with one or more amino acid residue(s) in a CD8+
T-cell epitope added to the polypeptide. In certain further
embodiments, the disrupting step further comprises the step or
steps of making one or more amino acid substitutions in the CD4+
T-cell epitope. In certain further embodiments, the disrupting step
further comprises the step or steps of making one or more amino
acid insertions in the CD4+ T-cell epitope.
[0051] In certain embodiments of the methods of the present
invention is a method for reducing CD4+ T-cell immunogenicity in a
polypeptide, the method comprising the steps of: identifying a CD4+
T-cell epitope in a polypeptide; and disrupting the identified CD4+
T-cell epitope with one or more amino acid residue(s) in a CD8+
T-cell epitope added to the polypeptide. In certain further
embodiments, the disrupting step further comprises the step or
steps of making one or more amino acid substitutions in the CD4+
T-cell epitope. In certain further embodiments, the disrupting step
further comprises the step or steps of making one or more amino
acid insertions in the CD4+ T-cell epitope.
[0052] In certain embodiments of the methods of the present
invention is a method for reducing CD4+ T-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity of the polypeptide, the method comprising the step
of: disrupting a CD4+ T-cell epitope with one or more amino acid
residue(s) in a heterologous CD8+ T-cell epitope added to the
polypeptide. In certain further embodiments, the disrupting step
further comprises the step or steps of making one or more amino
acid substitutions in the CD4+ T-cell epitope. In certain further
embodiments, the disrupting step further comprises the step or
steps of making one or more amino acid insertions in the CD4+
T-cell epitope.
[0053] In certain embodiments of the methods of the present
invention is a method for reducing CD4+ T-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity of the polypeptide, the method comprising the steps
of: identifying a CD4+ T-cell epitope in a polypeptide; and
disrupting the identified CD4+ T-cell epitope with one or more
amino acid residue(s) in a CD8+ T-cell epitope added to the
polypeptide. In certain further embodiments, the disrupting step
further comprises the step or steps of making one or more amino
acid substitutions in the CD4+ T-cell epitope. In certain further
embodiments, the disrupting step further comprises the step or
steps of making one or more amino acid insertions in the CD4+
T-cell epitope.
[0054] Certain embodiments of the polypeptides of the present
invention provide a polypeptide produced by any of the methods of
the present invention.
[0055] In certain embodiments, the polypeptide of the present
invention comprises or consists essentially of any one of SEQ ID
NOs: 11-43 or 46-48.
[0056] In certain embodiments, a cell-targeted molecule of the
present invention comprises a cell-targeting moiety or agent, and
any polypeptide of the present invention. In certain further
embodiments, the cell-targeted molecule further comprises a binding
region comprising one or more polypeptides and capable of
specifically binding at least one extracellular target biomolecule.
In certain further embodiments, the binding region comprises a
polypeptide selected from the group consisting of: a complementary
determining region 3 (CDR3) fragment constrained FR3-CDR3-FR4
(FR3-CDR3-FR4) polypeptide, single-domain antibody fragment (sdAb),
nanobody, heavy-chain antibody domain derived from a camelid
(V.sub.HH fragment), heavy-chain antibody domain derived from a
cartilaginous fish, immunoglobulin new antigen receptors (IgNARs),
V.sub.NAR fragment, single-chain variable fragment (scFv), antibody
variable fragment (Fv), antigen-binding fragment (Fab), Fd
fragment, small modular immunopharmaceutical (SMIP) domain,
fibronectin-derived 10.sup.th fibronectin type III domain (10Fn3)
(e.g. monobody), tenascin type III domain (e.g. TNfn3), ankyrin
repeat motif domain (ARD), low-density-lipoprotein-receptor-derived
A-domain (A domain of LDLR or LDLR-A), lipocalin (anticalin),
Kunitz domain, Protein-A-derived Z domain, gamma-B
crystalline-derived domain (Affilin), ubiquitin-derived domain,
Sac7d-derived polypeptide, Fyn-derived SH2 domain (affitin),
miniprotein, C-type lectin-like domain scaffold, engineered
antibody mimic, and any genetically manipulated counterparts of any
of the foregoing that retain binding functionality. In certain
further embodiments of the cell-targeted molecule of the present
invention, whereby upon administration of the cell-targeted
molecule to a cell physically coupled with an extracellular target
biomolecule of the binding region, the cell-targeted molecule is
capable of causing death of the cell. In certain further
embodiments of the cell-targeted molecule of the present invention,
whereby upon administration of the cell-targeted molecule to a
first population of cells whose members are physically coupled to
extracellular target biomolecules of the binding region, and a
second population of cells whose members are not physically coupled
to any extracellular target biomolecule of said binding region, the
cytotoxic effect of the cell-targeted molecule to members of said
first population of cells relative to members of said second
population of cells is at least 3-fold greater. In certain further
embodiments of the cell-targeted molecules of the present
invention, the binding region is capable of binding to an
extracellular target biomolecule selected from the group consisting
of: CD20, CD22, CD40, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR,
EpCAM, EphB2, prostate-specific membrane antigen, Cripto, endoglin,
fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7,
CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase
IX, folate binding protein, ganglioside GD2, ganglioside GD3,
ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3,
Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1,
TRAIL-R2, RANKL, FAP, tenascin, CD64, mesothelin, BRCA1,
MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,
GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1,
caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic
antigen, prostate specific antigen, prostate stem cell antigen,
human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3,
MUC1, MART-1/MelanA, gp100, tyrosinase associated antigen, HPV-E7,
Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen,
17-Al, bladder tumor antigen, CD38, CD15, CD23, CD53, CD88, CD129,
CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9,
mrp-14, Siglec-8, Siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69,
CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86,
CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC Class
II, CD284-TLR4, CD107-Mac3, CD195-CCR5, HLA-DR, CD16/32,
CD282-TLR2, CD11c, and any immunogenic fragment of any of the
foregoing. In certain further embodiments of the cell-targeted
molecules of the present invention, the cell-targeted molecule
further comprises a carboxy-terminal endoplasmic reticulum
retention/retrieval signal motif of a member of the KDEL family. In
certain further embodiments, the carboxy-terminal endoplasmic
reticulum retention/retrieval signal motif selected from the group
consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF,
HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL,
KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL,
HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL,
RTDL, RTEL, SDEL, TDEL, and SKEL.
[0057] In certain embodiments of the present invention, upon
administration of the cell-targeted molecule of the present
invention to a cell physically coupled with an extracellular target
biomolecule of the cell-targeting moiety of the cytotoxic protein,
the cytotoxic protein is capable of causing death of the cell.
[0058] In certain embodiments of the present invention, upon
administration of the cell-targeted molecule of the present
invention to two different populations of cell types with respect
to the presence of an extracellular target biomolecule, the
cell-targeted molecule is capable of causing cell death to the
cell-types physically coupled with an extracellular target
biomolecule of the cell-targeting moiety or agent's binding region
at a CD.sub.50 at least three times or less than the CD.sub.50 to
cell types which are not physically coupled with an extracellular
target biomolecule of the cell-targeted molecule's cell-targeting
moiety.
[0059] In certain embodiments, the cell-targeted molecule of the
present invention comprises or consists essentially of a
polypeptide of any one of the amino acid sequences of SEQ ID NOs:
49-60.
[0060] In certain further embodiments, the polypeptides of the
present invention comprise a mutation which reduces or eliminates
catalytic activity of a toxin-derived polypeptide but retains at
least one other toxin effector function. In certain embodiments,
the cell-targeted molecule of the present invention further
comprises a toxin effector polypeptide, derived from a toxin
effector polypeptide with enzymatic activity, which comprises a
mutation relative to a naturally occurring toxin which changes the
enzymatic activity of the toxin effector polypeptide. In certain
further embodiments, the mutation is selected from at least one
amino acid residue deletion, insertion, or substitution that
reduces or eliminates cytotoxicity of the toxin effector
polypeptide. In certain embodiments, the cell-targeted molecules of
the invention comprise a Shiga toxin effector region which further
comprises a mutation relative to a naturally occurring A Subunit of
a member of the Diphtheria toxin family that changes the enzymatic
activity of the diphtheria toxin effector region, the mutation
selected from at least one amino acid residue deletion or
substitution, such as, e.g. H21A, Y27A, W50A, Y54A, Y65A, E148A,
and W153A. In certain embodiments, the cell-targeted molecules of
the invention comprise a Shiga toxin effector region which further
comprises a mutation relative to a naturally occurring A Subunit of
a member of the Shiga toxin family that changes the enzymatic
activity of the Shiga toxin effector region, the mutation selected
from at least one amino acid residue deletion or substitution, such
as, e.g., A231E, R75A, Y77S, Y114S, E167D, R170A, R176K and/or
W203A in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
[0061] The present invention also provides pharmaceutical
compositions comprising a polypeptide and/or cell-targeted molecule
of the invention and at least one pharmaceutically acceptable
excipient or carrier; and the use of such a polypeptide,
cell-targeted molecule, or a composition comprising it in methods
of the invention as further described herein. Certain embodiments
of the present invention are pharmaceutical compositions comprising
any polypeptide of the present invention and/or any cell-targeted
molecule of the present invention; and at least one
pharmaceutically acceptable excipient or carrier.
[0062] Beyond the polypeptides, cell-targeted molecules, proteins,
and compositions of the present invention, polynucleotides capable
of encoding a polypeptide comprising a polypeptide or cell-targeted
molecule or protein of the present invention comprising a
polypeptide of the invention are within the scope of the present
invention, as well as expression vectors which comprise a
polynucleotide of the invention and host cells comprising an
expression vector of the invention. Host cells comprising an
expression vector may be used, e.g., in methods for producing a
polypeptide and/or protein of the invention comprising it, or a
polypeptide component or fragment thereof, by recombinant
expression.
[0063] Additionally, the present invention provides methods of
selectively killing cell(s) comprising the step of contacting a
cell(s) with a cell-targeted molecule of the invention or a
pharmaceutical composition comprising such a protein of the
invention. In certain embodiments, the step of contacting the
cell(s) occurs in vitro. In certain other embodiments, the step of
contacting the cell(s) occurs in vivo.
[0064] The present invention further provides methods of treating
diseases, disorders, and/or conditions in patients in need thereof
comprising the step of administering to a patient in need thereof a
therapeutically effective amount of a composition comprising a
polypeptide of the invention, a polypeptide and/or protein
comprising it, or a composition comprising any of the foregoing
(e.g., a pharmaceutical composition). In certain embodiments, the
disease, disorder, or condition to be treated using this method of
the invention is selected from: a cancer, tumor, immune disorder,
or microbial infection. In certain embodiments of this method, the
cancer to be treated is selected from the group consisting of: bone
cancer, breast cancer, central/peripheral nervous system cancer,
gastrointestinal cancer, germ cell cancer, glandular cancer,
head-neck cancer, hematological cancer, kidney-urinary tract
cancer, liver cancer, lung/pleura cancer, prostate cancer, sarcoma,
skin cancer, and uterine cancer. In certain embodiments of this
method, the immune disorder to be treated is an immune disorder
associated with a disease selected from the group consisting of:
amyloidosis, ankylosing spondylitis, asthma, Crohn's disease,
diabetes, graft rejection, graft-versus-host disease, Hashimoto's
thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus
erythematosus, multiple sclerosis, polyarteritis, psoriasis,
psoriatic arthritis, rheumatoid arthritis, scleroderma, septic
shock, Sjorgren's syndrome, ulcerative colitis, and vasculitis.
[0065] Among certain embodiments of the present invention is a
composition comprising a polypeptide of the invention, a
polypeptide and/or cell-targeted molecule comprising it, or a
composition comprising any of the foregoing, for the treatment or
prevention of a cancer, tumor, immune disorder, or microbial
infection. Among certain embodiments of the present invention is
the use of a composition of matter of the invention in the
manufacture of a medicament for the treatment or prevention of a
cancer, tumor, immune disorder, or microbial infection.
[0066] Certain embodiments of the cell-targeted molecules of the
present invention may be used to deliver one or more additional
exogenous materials into a cell physically coupled with an
extracellular target biomolecule of the protein of the present
invention. Additionally, the present invention provides a method
for delivering exogenous material to the inside of a cell(s)
comprising contacting the cell(s), either in vitro or in vivo, with
a cell-targeted molecule, pharmaceutical composition, and/or
diagnostic composition of the present invention. The present
invention further provides a method for delivering exogenous
material to the inside of a cell(s) in a patient in need thereof,
the method comprising the step of administering to the patient a
cell-targeted molecule of the present invention, wherein the target
cell(s) is physically coupled with an extracellular target
biomolecule of the protein of the present invention.
[0067] Among certain embodiments of the present invention is the
use of a compound (e.g. a polypeptide or a cell-targeted molecule)
of the invention and/or composition (e.g. a pharmaceutical
composition) of the invention in the diagnosis, prognosis, or
characterization of a disease, disorder, or condition.
[0068] Among certain embodiments of the present invention is a
diagnostic composition comprising a polypeptide of the invention
and/or cell-targeted molecule comprising it, or a composition
comprising any of the foregoing, and a detection promoting agent
for the collection of information, such as diagnostically useful
information about a cell type, tissue, organ, disease, disorder,
condition, or patient.
[0069] Among certain embodiments of the present invention is the
method of detecting a cell using a cell-targeted molecule and/or
diagnostic composition of the invention comprising the steps of
contacting a cell with said cell-targeted molecule and/or
diagnostic composition and detecting the presence of said
cell-targeted molecule and/or diagnostic composition. In certain
embodiments, the step of contacting the cell(s) occurs in vitro. In
certain embodiments, the step of contacting the cell(s) occurs in
vivo. In certain embodiments, the step of detecting the cell(s)
occurs in vitro. In certain embodiments, the step of detecting the
cell(s) occurs in vivo.
[0070] For example, a diagnostic composition of the invention may
be used to detect a cell in vivo by administering to a mammalian
subject a composition comprising protein of the present invention
which comprises a detection promoting agent and then detecting the
presence of the protein of the present invention either in vitro or
in vivo. The information collected may regard the presence of a
cell physically coupled with an extracellular target of the binding
region of the cell-targeted molecule of the present invention and
may be useful in the diagnosis, prognosis, characterization, and/or
treatment of a disease, disorder, or condition. Certain compounds
(e.g. polypeptides and cell-targeted molecules) of the invention,
compositions (e.g. pharmaceutical compositions and diagnostic
compositions) of the invention, and methods of the invention may be
used to determine if a patient belongs to a group that responds to
a pharmaceutical composition of the invention.
[0071] Certain embodiments of the polypeptides of the present
invention and the cell-targeted molecules of the present invention
may be utilized as an immunogen or as a component of an immunogen
for the immunization and/or vaccination of a chordate.
[0072] For certain embodiments, a method of the invention is for
"seeding" a tissue locus within a chordate, the method comprising
the step of: administering to the chordate a cell-targeted molecule
of the invention, a pharmaceutical composition of the invention, or
a diagnostic composition of the invention. In certain further
embodiments, the methods of the invention for "seeding" a tissue
locus are for "seeding" a tissue locus which comprises a malignant,
diseased, or inflamed tissue. In certain further embodiments, the
methods of the invention for "seeding" a tissue locus are for
"seeding" a tissue locus which comprises the tissue selected from
the group consisting of: diseased tissue, tumor mass, cancerous
growth, tumor, infected tissue, or abnormal cellular mass. In
certain further embodiments, the methods of the invention for
"seeding" a tissue locus comprises administering to the chordate
the cell-targeted molecule of the invention, the pharmaceutical
composition of the invention, or the diagnostic composition of the
invention comprising the heterologous T-cell epitope selected from
the group consisting of: peptides not natively presented by the
target cells of the cell-targeted molecule in MHC class I
complexes, peptides not natively present within any protein
expressed by the target cell, peptides not natively present within
the proteome of the target cell, peptides not natively present in
the extracellular microenvironment of the site to be seeded, and
peptides not natively present in the tumor mass or infected tissue
site to be targeted.
[0073] Among certain embodiments of the present invention are kits
comprising a composition of matter of the present invention, and
optionally, instructions for use, additional reagent(s), and/or
pharmaceutical delivery device(s).
[0074] These and other features, aspects and advantages of the
present invention will become better understood with regard to the
following description, appended claims, and accompanying figures.
The aforementioned elements of the invention may be individually
combined or removed freely in order to make other embodiments of
the invention, without any statement to object to such combination
or removal hereinafter.
BRIEF DESCRIPTION OF THE FIGURES
[0075] FIG. 1 shows the general arrangement of exemplary T-cell
epitope presenting effector polypeptides, including B-cell/CD4+
T-cell de-immunized variants, and cell-targeted proteins comprising
the same.
[0076] FIG. 2 shows embedding T-cell epitopes into B-cell epitope
regions of a toxin effector polypeptide did not significantly
impair catalytic activity. Two exemplary Diphtheria toxin-derived
polypeptides comprising a T-cell epitope embedded into a B-cell
epitope region exhibited levels of ribosome inactivation comparable
to a wild-type Diphtheria toxin.
[0077] FIG. 3 shows embedding or inserting T-cell epitopes into a
B-cell epitope region disrupted epitope(s) recognized by various
anti-SLT-1A antibodies by Western blot analysis.
[0078] FIG. 4 shows embedding T-cell epitopes into various B-cell
epitope regions disrupted epitope(s) recognized by various
anti-SLT-1A antibodies by Western blot analysis.
[0079] FIG. 5 shows overlays of the results of flow cytometric
analysis of sets of cells receiving different treatments:
untreated, treated with an exemplary cell-targeted protein of the
present invention, treated with exogenous epitope-peptide and PLE,
and treated with exogenous epitope-peptide only. Cells treated with
three exemplary cell-targeted proteins of the present invention,
each comprising a de-immunized Shiga toxin effector polypeptide
comprising an embedded T-cell epitope disrupting a B-cell epitope
region, displayed the embedded epitope-peptide complexed to MHC
molecules on their cell surfaces.
DETAILED DESCRIPTION
[0080] The present invention is described more fully hereinafter
using illustrative, non-limiting embodiments, and references to the
accompanying figures. This invention may, however, be embodied in
many different forms and should not be construed as to be limited
to the embodiments set forth below. Rather, these embodiments are
provided so that this disclosure is thorough and conveys the scope
of the invention to those skilled in the art.
[0081] In order that the present invention may be more readily
understood, certain terms are defined below. Additional definitions
may be found within the detailed description of the invention.
[0082] As used in the specification and the appended claims, the
terms "a," "an" and "the" include both singular and the plural
referents unless the context clearly dictates otherwise.
[0083] As used in the specification and the appended claims, the
term "and/or" when referring to two species, A and B, means at
least one of A and B. As used in the specification and the appended
claims, the term "and/or" when referring to greater than two
species, such as A, B, and C, means at least one of A, B, or C, or
at least one of any combination of A, B, or C (with each species in
singular or multiple possibility).
[0084] Throughout this specification, the word "comprise" or
variations such as "comprises" or "comprising" will be understood
to imply the inclusion of a stated integer (or components) or group
of integers (or components), but not the exclusion of any other
integer (or components) or group of integers (or components).
[0085] Throughout this specification, the term "including" is used
to mean "including but not limited to." "Including" and "including
but not limited to" are used interchangeably.
[0086] The term "amino acid residue" or "amino acid" includes
reference to an amino acid that is incorporated into a protein,
polypeptide, or peptide. The term "polypeptide" includes any
polymer of amino acids or amino acid residues. The term
"polypeptide sequence" refers to a series of amino acids or amino
acid residues which physically comprise a polypeptide. A "protein"
is a macromolecule comprising one or more polypeptides or
polypeptide "chains". A "peptide" is a small polypeptide of sizes
less than a total of 15-20 amino acid residues. The term "amino
acid sequence" refers to a series of amino acids or amino acid
residues which physically comprise a peptide or polypeptide
depending on the length. Unless otherwise indicated, polypeptide
and protein sequences disclosed herein are written from left to
right representing their order from an amino terminus to a carboxy
terminus.
[0087] The terms "amino acid," "amino acid residue," "amino acid
sequence," or polypeptide sequence include naturally occurring
amino acids and, unless otherwise limited, also include known
analogs of natural amino acids that can function in a similar
manner as naturally occurring amino acids, such as selenocysteine,
pyrrolysine, N-formylmethionine, gamma-carboxyglutamate,
hydroxyprolinehypusine, pyroglutamic acid, and selenomethionine.
The amino acids referred to herein are described by shorthand
designations as follows in Table A:
TABLE-US-00001 TABLE A Amino Acid Nomenclature Name 3-letter
1-letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic
Acid or Aspartate Asp D Cysteine Cys C Glutamic Acid or Glutamate
Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile
I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F
Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W
Tyrosine Tyr Y Valine Val V
[0088] The phrase "conservative substitution" with regard to a
polypeptide, refers to a change in the amino acid composition of
the polypeptide that does not substantially alter the function and
structure of the overall polypeptide (see Creighton, Proteins:
Structures and Molecular Properties (W. H. Freeman and Company, New
York (2nd ed., 1992)).
[0089] As used herein, the term "expressed," "expressing" or
"expresses" refers to translation of a polynucleotide or nucleic
acid into a polypeptide or protein. The expressed polypeptide or
proteins may remain intracellular, become a component of the cell
surface membrane or be secreted into an extracellular space.
[0090] As used herein, the symbol ".alpha." is shorthand for an
immunoglobulin-type binding region capable of binding to the
biomolecule following the symbol. The symbol ".alpha." is used to
refer to the functional characteristic of an immunoglobulin-type
binding region based on its capability of binding to the
biomolecule following the symbol.
[0091] The symbol "::" means the polypeptide regions before and
after it are physically linked together to form a continuous
polypeptide.
[0092] For purposes of the present invention, the phrase "derived
from" means that the polypeptide region comprises amino acid
sequences originally found in a protein and which may now comprise
additions, deletions, truncations, or other alterations relative to
the original sequence such that overall function and structure are
substantially conserved.
[0093] For purposes of the present invention, the term "effector"
means providing a biological activity, such as cytotoxicity,
biological signaling, enzymatic catalysis, subcellular routing,
and/or intermolecular binding resulting in the recruitment one or
more factor(s), and/or allosteric effects.
[0094] As used herein, the terms "subunit" and "chain" with regard
to multimeric toxins, such as, e.g., ABx toxins, are used
interchangeably.
[0095] For purposes of the present invention, the phrase "CD8+
T-cell hyper-immunized" means that the molecule, when present
inside a nucleated, chordate cell within a living chordate, has an
increased antigenic and/or immunogenic potential regarding CD8+
T-cell antigenicity or immunogenicity. Commonly, CD8+ T-cell
immunized molecules are capable of cellular internalization to an
early endosomal compartment of a nucleated, chordate cell due
either to an inherent feature(s) or as a component of a
cell-targeted molecule.
[0096] For purposes of the present invention, the phrase "B-cell
and/or CD4+ T-cell de-immunized" means that the molecule has a
reduced antigenic and/or immunogenic potential after administration
to a mammal regarding either B-cell antigenicity or immunogenicity
and/or CD4+ T-cell antigenicity or immunogenicity.
[0097] For purposes of the present invention, the term "proteasome
delivering effector" means a molecule that provides the biological
activity of localizing within a cell to a subcellular compartment
that is competent to result in the proteasomal degradation of the
proteasome delivering effector molecule. Generally, this proteasome
delivering biological activity can be determined from the initial
sub-cellular location of the proteasome delivering effector
molecule in an early endosomal compartment; however, it can also be
determined earlier, e.g., from an extracellular starting location
which involves passage into a cell and through an endosomal
compartment of the cell, such as, e.g. after endocytotic entry into
that cell. Alternatively, proteasome delivering effector biological
activity may in certain embodiments not involve passage through any
endosomal compartment of a cell before the proteasome delivering
effector polypeptide is internalized and reaches a compartment
competent to result in its proteasomal degradation. The ability of
a given molecule to provide proteasome delivering effector
function(s) may be assayed by the skilled worker using techniques
known in the art.
[0098] The term "heterologous" with regard to T-cell epitope or
T-cell epitope peptide component of a polypeptide of the present
invention refers to an epitope or peptide sequence which did not
initially occur in the polypeptide to be modified, but which has
been added to the polypeptide using a method of the present
invention, whether added via the processes of embedding, fusion,
insertion, and/or amino acid substitution as described herein, or
by any other engineering means. The result is a modified
polypeptide comprising a T-cell epitope foreign to the original,
unmodified polypeptide, i.e. the T-cell epitope was not present in
the original polypeptide.
[0099] The term "endogenous" with regard to a B-cell epitope or
CD4+ T-cell epitope in a polypeptide refers to an epitope already
present in the polypeptide before being modified by a method of the
present invention.
[0100] As used herein, the terms "disrupted", "disruption," or
"disrupting," with regard to a polypeptide region or feature within
a polypeptide refers to an alteration of at least one amino acid
within the region or composing the feature. Amino acid alterations
include various mutations, such as, e.g., a deletion, inversion,
insertion, or substitution which alter the amino acid sequence of
the polypeptide. Amino acid alterations also include chemical
changes, such as, e.g., the alteration one or more atoms in an
amino acid functional group or the addition of one or more atoms to
an amino acid functional group.
[0101] The phrase "in association with" or "associated with" with
regard to a T-cell epitope or T-cell epitope peptide component of a
polypeptide of the present invention means the T-cell epitope and
polypeptide are physically linked together, whether by covalent or
non-covalent linkages, such as, e.g., embedded or inserted within
the polypeptide, fused to the polypeptide, and/or chemically
conjugated to the polypeptide.
[0102] The term "associating" with regard to the claimed invention
means the act of making two molecules associated with each other or
in association with each other.
[0103] The term "embedded" and grammatical variants thereof, with
regard to a T-cell epitope or T-cell epitope peptide component of a
polypeptide of the present invention refers to the internal
replacement of one or more amino acids within a polypeptide region
with different amino acids in order to generate a new polypeptide
sequence sharing the same total number of amino acid residues.
Thus, the term embedded does not include any external, terminal
fusion of any additional amino acid, peptide, or polypeptide
component to the starting polypeptide nor any additional internal
insertion of any additional amino acid residues but rather only
substitutions for existing amino acids. The internal replacement
may be accomplished merely by amino acid residue substitution or by
a series of substitutions, deletions, insertions, and/or
inversions. If an insertion of one or more amino acids is used,
then the equivalent number of proximal amino acids must be deleted
next to the insertion to result in an embedded T-cell epitope. This
is in contrast to use of the term "inserted" with regard to T-cell
epitopes in the polypeptides of the present invention which instead
refers to a polypeptide with an increased length equivalent to the
length of the inserted T-cell epitope. Insertions include the
previous even if other regions of the polypeptide not proximal to
the insertion are deleted to then decrease the total length of the
final polypeptide.
[0104] The term "fused" and grammatical variants thereof, with
regard to a T-cell epitope or T-cell epitope peptide component of a
polypeptide of the present invention refers to the external
addition of four, five, six, or more amino acids to either the
amino-terminus or carboxy terminus of a polypeptide in order to
generate a new polypeptide which has a greater number of amino acid
residues than the original. Fused T-cell epitopes include the
addition of four, five, six, or more amino acids to either the
amino-terminus or carboxy terminus even if other regions of the
polypeptide are deleted to then decrease the total length of the
final polypeptide as long as the new polypeptide retains an
effector function of the original polypeptide, such as, e.g.,
proteasome delivering effector function.
[0105] As used herein, the term "toxin effector polypeptide" means
a polypeptide that comprises a toxin-derived effector region that
is sufficient to provide one or more biological activities present
in the toxin from which the polypeptide was derived.
[0106] As used herein, the term "T-cell epitope delivering" means
that a molecule provides the biological activity of localizing
within a cell to a subcellular compartment that is competent to
result in the proteasomal degradation of a T-cell epitope carrying
polypeptide region. Generally, this proteasome delivering
biological activity can be determined from the initial sub-cellular
location of the T-cell epitope delivering molecule in an early
endosomal compartment; however, it can also be determined earlier,
e.g., from an extracellular starting location which involves
passage into a cell and through an endosomal compartment of the
cell, such as, e.g. after endocytotic entry into that cell.
Alternatively, T-cell epitope delivering activity may in certain
embodiments not involve passage through any endosomal compartment
of a cell before the T-cell epitope delivering molecule is
internalized and reaches a compartment competent to deliver a
T-cell epitope to the proteasome for degradation into a T-cell
epitope peptide. Effective T-cell epitope delivering function can
be assayed by observing MHC presentation of the delivered T-cell
epitope on a cell surface of a cell in which the T-cell epitope
delivering molecule has internalized.
[0107] As used herein, a toxin effector function or activity may
include, inter alia, promoting cellular internalization, promoting
endosome escape, directing intracellular routing to a subcellular
compartment, catalytic functions, substrate binding, inducing
apoptosis of cell, causing cytostasis, and cytotoxicity.
[0108] As used herein, the retention of a toxin-derived polypeptide
effector function refers to a level of toxin effector functional
activity, as measured by an appropriate quantitative assay with
reproducibility, comparable to a wild-type polypeptide (WT)
control. For example, various assays know to the skilled worker may
be used to measure the enzymatic activity and/or intracellular
routing of a toxin effector polypeptide. The enzymatic polypeptide
effector toxin function of a polypeptide of the present invention
is retained if its enzymatic activity is comparable to a wild-type
polypeptide (WT) in the same assay under the same conditions.
[0109] The term "selective cytotoxicity" with regard to the
cytotoxic activity of a cytotoxic protein refers to the relative
levels of cytotoxicity between a targeted cell population and a
non-targeted bystander cell population, which can be expressed as a
ratio of the half-maximal cytotoxic concentration (CD.sub.50) for a
targeted cell type over the CD.sub.50 for an untargeted cell type
to show preferentiality of cell killing of the targeted cell
type.
INTRODUCTION
[0110] The present invention provides methods of generating
polypeptides and cell-targeted molecules which are capable of
delivering T-cell epitope peptides to the MHC class I system of a
target cell for cell surface presentation. The present invention
also provides exemplary T-cell epitope delivering polypeptides,
made using the methods of the invention, which are capable of
delivering heterologous T-cell epitopes to the MHC class I system
of a target cell for cell surface presentation. The polypeptides
created using the methods of the present invention, e.g. T-cell
epitope delivering polypeptides and CD8+ T-cell hyper-immunized
polypeptides, may be utilized as components of various molecules
and compositions, e.g. cytotoxic therapeutics, therapeutic delivery
agents, and diagnostic molecules.
[0111] In addition, the present invention provides methods of
generating variants of polypeptides by simultaneously reducing the
probability of B-cell antigenicity and/or immunogenicity while
providing at an overlapping position within the polypeptide a
heterologous T-cell epitope for increasing the probability of
T-cell immunogenicity via MHC class I presentation. The present
invention also provides exemplary B-cell epitope de-immunized,
T-cell epitope delivering polypeptides made using the methods of
the invention, which are capable of delivering heterologous T-cell
epitopes to the MHC class I system of a target cell for cell
surface presentation. The polypeptides created using the methods of
the present invention, e.g. B-cell/CD4+ T-cell de-immunized T-cell
epitope delivering polypeptides, CD8+ T-cell hyper-immunized and
CD4+ T-cell de-immunized polypeptides, may be utilized as
components of various molecules and compositions, e.g. cytotoxic
therapeutics, therapeutic delivery agents, and diagnostic
molecules.
I. The General Structure of a CD8+ T-Cell Hyper-Immunized
Polypeptide
[0112] The present invention involves the engineering of
polypeptides comprising various proteasome delivery effector
polypeptide regions to comprise one or more heterologous T-cell
epitopes; and where upon delivery of the polypeptide to an early
endosomal compartment of a eukaryotic cell, the polypeptide is
capable of localizing within the cell to a subcellular compartment
sufficient for delivering the one or more heterologous T-cell
epitopes to the proteasome for degradation and entry into the
cell's MHC class I system. While the proteasome delivery effector
polypeptides may come from any source, in certain embodiments, the
polypeptides of the invention are derived from various proteasome
delivery effector polypeptides derived from naturally occurring
protein toxins.
A. Polypeptides Engineered to Comprise One or More Heterologous.
T-Cell Epitopes and a Proteasome Delivery Effector Polypeptide
[0113] The present invention contemplates the use of various
polypeptides as a starting point for modification into a
polypeptide of the present invention via the embedding, fusing,
and/or inserting of one or more heterologous T-cell epitopes. These
source polypeptides should exhibit, or be predicted, to exhibit a
proteasome delivery effector capability.
[0114] The predominant source of peptide epitopes entering the MHC
class I pathway are peptides resulting from proteasomal degradation
of cytosolic molecules. However, ER-localized molecules, such as
viral glycoproteins and transformed cell glycoproteins, can also be
displayed by the MHC class I system by a different route. Although
this alternative route to MHC class I presentation begins in the
ER, the polypeptide or protein source of the peptide is transported
to the cytosol for proteloytic processing by the proteasome before
being transported by TAPs to the lumen of the ER for peptide
loading onto MHC class I molecules. The exact mechanism underlying
this alternative route is not clear but might involve an
ER-associated degradation (ERAD)-type surveillance system to detect
misfolded proteins, "defective ribosomal products," and structures
mimicking the aforementioned. This ERAD-type system transports
certain polypeptides and proteins to proteasomes in the cytosol for
degradation, which can result in the production of cytosolic
antigenic peptides. In addition, research on the intracellular
routing of various toxins suggests that reaching either the cytosol
or the endoplasmic reticulum is sufficient for delivery of a T-cell
epitope into the MHC class I pathway.
[0115] Therefore, polypeptides and proteins known or discovered to
localize to and/or direct their own intracellular transport to the
cytosol and/or endoplasmic reticulum represent a class of molecules
predicted to comprise one or more proteasome delivery effector
polypeptide regions which exhibit a proteasome delivery
function(s). Certain proteins and polypeptides, such as, e.g.,
certain toxins, exhibit the ability to escape from endosomal
compartments into the cytosol, thereby avoiding lysosomal
degradation. Thus, polypeptides and proteins known or discovered to
escape endosomal compartments and reach the cytosol are included in
the class of molecules mentioned above. The exact route the
polypeptide or protein takes to the cytosol or ER is irrelevant as
long as the polypeptide or protein eventually reaches a subcellular
location that permits access to the proteasome.
[0116] In addition, certain molecules are able to reach the
proteasome of a cell after being localized to a lysosome. For
example, foreign proteins introduced directly into the cytosol of a
cell, such as listeriolysin and other proteins secreted by Listeria
monocytogenes, can enter the MHC class I pathway and be presented
in MHC class I complexes for recognition by effector T-cells
(Villanueva M et al., J Immunol 155: 5227-33 (1995)). In addition,
lysosomal proteolysis, including phagolysosome proteolysis, can
produce antigenic peptides that are translocated into the cytosol
and enter the MHC class I pathway for cell surface presentation in
a process called cross-presentation, which may have evolved from a
canonical ERAD system (Gagnon E et al., Cell 110: 119-31 (2002)).
Thus, certain polypeptides and proteins known or discovered to
localize to lysosomes may be suitable sources for polypeptides with
proteasome delivery effector regions which exhibit a proteasome
delivery function(s).
[0117] The ability of a proteinaceous molecule to intracellularly
route to the cytosol, ER, and/or lysosomal compartments of a cell
from the starting position of an early endosomal compartment can be
determined by the skilled worker using assays known in the art.
Then, the proteasome delivery effector polypeptide regions of a
source polypeptide or protein, such as, e.g., a toxin, can be
mapped and isolated by the skilled worker using standard techniques
known in the art.
1. Proteasome Delivery Effector Polypeptides Derived from
Toxins
[0118] The present invention contemplates the use of various
polypeptides derived from toxins as proteasome delivering effector
regions. Many toxins represent optimal sources of proteasome
delivering effector polypeptides because of the wealth of knowledge
about their intracellular routing behaviors.
[0119] Many naturally occurring proteinaceous toxins have highly
evolved structures optimized for directing intracellular routing in
vertebrate host cells, including via endosomal escape and
retrograde transport pathways.
[0120] Numerous toxins exhibit endosome escape properties, commonly
via pore formation (Mandal M et al., Biochim Biophys Acta 1563:
7-17 (2002)). For example, diphtheria toxin and plant type II
ribosome inactivating proteins like ricin can escape from endosomes
(Murphy S et al., Biochim Biophys Acta 1824: 34-43 (2006);
Slominska-Wojewodzka M, Sandvig K, Antibodies 2: 236-269 (2013);
Walsh M et al., Virulence 4: 774-84 (2013)). Escape from endosomal
compartments, including lysosomes, can be measured directly and
quantitated using assays known in the art, such as, e.g., using
reporter assays with horseradish peroxidase, bovine serum albumin,
fluorophores like Alexa 488, and toxin derived polypetides (see
e.g. Bartz R et al., Biochem J 435: 475-87 (2011); Gilabert-Oriol R
et al., Toxins 6: 1644-66 (2014)).
[0121] Many toxins direct their own intracellular routing in
vertebrate host cells. For example, the intoxication pathways of
many toxins can be described as a multi-step process involving 1)
cellular internalization of the toxin into host cells, 2)
intracellular routing of the toxin via one or more sub-cellular
compartments, and 3) subsequent localization of a catalytic portion
of the toxin to the cytosol where host factor substrates are
enzymatically modified. For example, this process describes the
intoxication pathway of anthrax lethal factors, cholera toxins,
diphtheria toxins, pertussis toxins, Pseudomonas exotoxins, and
type II ribosome inactivating proteins like ricin and Shiga
toxins.
[0122] Similarly, recombinant toxins, modified toxin structures,
and engineered polypeptides derived from toxins can preserve these
same properties. For example, engineered recombinant polypeptides
derived from diphtheria toxin (DT), anthrax lethal factor (LF)
toxin, and Pseudomonas exotoxin A (PE) have been used as delivery
vehicles for moving polypeptides from an extracellular space to the
cytosol. Any protein toxin with the intrinsic ability to
intracellularly route from an early endosomal compartment to either
the cytosol or the ER represents a source for a proteasome delivery
effector polypeptide which may be exploited for the purposes of the
present invention, such as a starting component for modification or
as a source for mapping a smaller proteasome delivery effector
region therein.
[0123] For many toxins targeting eukaryotic cells, toxicity is the
result of an enzymatic mechanism involving a substrate(s) in the
cytosol (see Table I). These toxins have evolved toxin structures
with the ability to deliver enzymatically active polypeptide
regions of their holotoxins to the cytosol. The enzymatic regions
of these toxins may be used as starting components for creating the
polypeptides of the present invention.
TABLE-US-00002 TABLE I Exemplary Protein Toxin Sources of
Proteasome Delivering Effector Polypeptides Protein Toxin
Substrate-Subcellular Location Abrins sarcin-ricin loop-cytosol
Anthrax lethal factor MAPKK-cytosol Aspf1 sarcin-ricin loop-cytosol
Bouganin sarcin-ricin loop-cytosol Bryodins sarcin-ricin
loop-cytosol Cholix toxin heterotrimeric G protein-cytosol
Cinnamomin sarcin-ricin loop-cytosol Claudin sarcin-ricin
loop-cytosol Clavin sarcin-ricin loop-cytosol C. difficile TcdA Ras
GTPases-cytosol C. difficile TcdA Rho GTPases-cytosol C.
perfringens iota Rho GTPases-cytosol cytolethal distending
DNA-nucleus Dianthins sarcin-ricin loop-cytosol Diphtheria toxins
elongation factor-2 (EF2)-cytosol Ebulins sarcin-ricin loop-cytosol
Gelonin sarcin-ricin loop-cytosol Gigantin sarcin-ricin
loop-cytosol heat-labile enterotoxins heterotrimeric G
protein-cytosol Maize RIPs sarcin-ricin loop-cytosol Mitogillin
sarcin-ricin loop-cytosol Nigrins sarcin-ricin loop-cytosol
Pertussis toxins heterotrimeric G protein-cytosol PD-Ls
sarcin-ricin loop-cytosol PAPs sarcin-ricin loop-cytosol
Pseudomonas toxins elongation factor-2 (EF2)-cytosol Pulchellin
sarcin-ricin loop-cytosol Restrictocin sarcin-ricin loop-cytosol
Ricins sarcin-ricin loop-cytosol Saporins sarcin-ricin loop-cytosol
Sarcins sarcin-ricin loop-cytosol Shiga toxins sarcin-ricin
loop-cytosol Subtilase cytotoxins endoplasmic chaperon-ER
Trichosanthins sarcin-ricin loop-cytosol
[0124] The toxins in two toxin superfamilies, with overlapping
members, are very amenable for use in the present invention: ABx
toxins and ribotoxins.
[0125] ABx toxins are capable of entering eukaryotic cells and
routing to the cytosol to attack their molecular targets.
Similarly, ribotoxins are capable of entering eukaryotic cells and
routing to the cytosol to inactivate ribosomes. Members of both the
Abx toxin and ribotoxin superfamilies are appropriate sources for
identifying toxin-derived polypeptides and proteasome delivery
effector polypeptides for use in the present invention
[0126] ABx toxins, which are also referred to as binary toxins, are
found in bacteria, fungi, and plants. The ABx toxins form a
superfamily of toxins that share the structural organization of two
or more polypeptide chains with distinct functions, referred to as
A and B subunits. The x represents the number of B subunits in the
holotoxins of the members of the ABx family, such as, e.g.,
AB.sub.1 for diphtheria toxin and AB.sub.5 for Shiga toxin. The AB5
toxin superfamily is comprised of 4 main families: cholix toxins
(Ct or Ctx), pertussis toxins (Ptx), Shiga toxins (Stx), and
Subtilase cytotoxins (SubAB). The cytotoxic mechanisms of AB5
toxins involves subcellular routing of their A subunits within an
intoxicated, eukaryotic, host cell to either the cytosol or the ER
where the catalytic A subunits act upon their enzymatic substrates
representing various host cell proteins (see Table I).
[0127] Diphtheria toxins disrupt proteins synthesis via the
catalytic ADP-ribosylation of the eukaryotic elongation factor-2
(EF2). Diphtheria toxins consists of a catalytic A subunit and a B
subunit, which contains a phospholipid bilayer translocation
effector domain and a cell-targeting binding domain. During the
diphtheria toxin intoxication process, diphtheria toxins can
intracellularly route their catalytic domains to the cytosol of a
eukaryotic cell, perhaps via endosomal escape (Murphy J, Toxins
(Basel) 3: 294-308 (2011)). This endosomal escape mechanism may be
shared with other toxins such as, e.g., anthrax lethal and edema
factors, and the general ability of endosome escape is exhibited by
many diverse toxins, including, e.g., certain C. difficile toxins,
gelonin, lysteriolysin, PE, ricin, and saporin (see e.g. Varkouhi A
et al., J Control Release 151: 220-8 (2010); Murphy J, Toxins
(Basel) 3: 294-308 (2011)).
[0128] In particular, toxins which inactivate ribosomes in the
cytosol are useful for identifying proteasome delivery effector
polypeptides for use in the present invention. These toxins
comprise polypeptide regions which simultaneously provide both
cytosol targeting effector function(s) and cytotoxic ribotoxic
toxin effector function(s).
[0129] With regard to the claimed invention, the phrase "ribotoxic
toxin effector polypeptide" refers to a polypeptide derived from
proteins, including naturally occurring ribotoxins and synthetic
ribotoxins, which is capable of effectuating ribosome inactivation
in vitro, protein synthesis inhibition in vitro and/or in vivo,
cytotoxicity, and/or cytostasis. Commonly, ribotoxic toxin effector
polypeptides are derived from naturally occurring protein toxins or
toxin-like structures which are altered or engineered by human
intervention. However, other polypeptides, such as, e.g., naturally
occurring enzymatic domains not natively present in a toxin or
synthetic polypeptide, are within the scope of that term as used
herein (see e.g. Newton D et al., Blood 97: 528-35 (2001); De
Lorenzo C et al., FEBS Lett 581: 296-300 (2007); De Lorenzo C,
D'Alessio G, Curr Pharm Biotechnol 9: 210-4 (2008); Menzel C et
al., Blood 111: 3830-7 (2008)). Thus, ribotoxic toxin effector
polypeptides may be derived from synthetic or engineered protein
constructs with increased or decreased ribotoxicity, and/or
naturally occurring proteins that have been otherwise altered to
have a non-native characteristic.
[0130] The ribotoxic toxin effector polypeptides may be derived
from ribotoxic domains of proteins from diverse phyla, such as,
e.g., algae, bacteria, fungi, plants, and animals. For example,
polypeptides derived from various ribotoxins have been linked or
fused to immunoglobulin domains or receptor ligands through
chemical conjugation or recombinant protein engineering with the
hope of creating cell-type-specific cytotoxic therapeutics (Pastan
I et al., Annu Rev Biochem 61: 331-54 (1992); Foss F et al., Curr
Top Microbiol Immunol 234: 63-81 (1998); Olsnes S, Toxicon 44:
361-70 (2004); Pastan I, et al., Nat Rev Cancer 6: 559-65 (2006);
Lacadena J et al., FEMS Microbiol Rev 31: 212-37 (2007); de
Virgilio M et al., Toxins 2: 2699-737 (2011); Walsh M, Virulence 4:
774-84 (2013); Weidle U et al., Cancer Genomics Proteomics 11:
25-38 (2014)).
[0131] Ribotoxic toxin effector polypeptides may be derived from
the catalytic domains of members of the Ribosome Inactivating
Protein (RIP) Superfamily of protein ribotoxins (de Virgilio M et
al., Toxins 2: 2699-737 (2011); Lapadula W et al., PLoS ONE 8:
e72825 (2013); Walsh M, Virulence 4: 774-84 (2013)). RIPs are
ribotoxic proteins expressed in algae, bacteria, fungi, and plants
which are often potent inhibitors of eukaryotic and prokaryotic
protein synthesis at sub-stoichiometric concentrations (see Stirpe,
F, Biochem J 202: 279-80 (1982)). Various RIPs are considered
promising sources for toxin effector polypeptide sequences for use
in therapeutics for treating cancers (see Pastan I, et al., Nat Rev
Cancer 6: 559-65 (2006); Fracasso G et al., Ribosome-inactivating
protein-containing conjugates for therapeutic use, Toxic Plant
Proteins 18, pp. 225-63 (Eds. Lord J, Hartley, M. Berlin,
Heidelberg: Springer-Verlag, 2010); de Virgilio M et al., Toxins 2:
2699-737 (2011); Puri M et al., Drug Discov Today 17: 774-83
(2012); Walsh M, Virulence 4: 774-84 (2013)).
[0132] The most commonly used ribotoxins in recombinant cytotoxic
polypeptides include diphtheria toxin, Pseudomonas exotoxin A,
ricin, .alpha.-sarcin, saporin, and gelonin (see Shapira A, Benhar
I, Toxins 2: 2519-83 (2010); Yu C et al., Cancer Res 69: 8987-95
(2009); Fuenmayor J, Montano R, Cancers 3: 3370-93 (2011); Weldon,
FEBS J 278: 4683-700 (2011); Carreras-Sangra N et al., Protein Eng
Des Sel 25: 425-35 (2012); Lyu M at al., Methods Enzymol 502:
167-214 (2012); Antignani, Toxins 5: 1486-502 (2013); Lin H et al.,
Anticancer Agents Med Chem 13: 1259-66 (2013); Polito L et al.,
Toxins 5: 1698-722 (2013); Walsh M, Virulence 4: 774-84 (2013)).
These ribotoxins are generally classified as ribosome inactivating
proteins (RIPs) and share a general cytotoxic mechanism of
inactivating eukaryotic ribosomes by attacking the sarcin-ricin
loop (SRL) or proteins required for ribosome function which bind to
the SRL.
[0133] The SRL structure is highly conserved between the three
phylogenetic groups, Archea, Bacteria and Eukarya, such that both
prokaryotic and eukaryotic ribosomes share a SRL ribosomal
structure (Gutell R et al., Nucleic Acids Res 21: 3055-74 (1993);
Szewczak A, Moore P, J Mol Biol 247: 81-98 (1995); Gluck A, Wool I,
J Mol Biol 256: 838-48 (1996); Seggerson K, Moore P, RNA 4: 1203-15
(1998); Correll C et al., J Mol Biol 292: 275-87 (1999)). The SRL
of various species from diverse phyla can be superimposed onto a
crystal structure electron density map with high precision (Ban N
et al., Science 11: 905-20 (2000); Gabashvili I et al., Cell 100:
537-49 (2000)). The SRL is the largest universally conserved
ribosomal sequence which forms a conserved secondary structure
vital to the ribosome function of translocation via the cooperation
of elongation factors, such as EF-Tu, EF-G, EF1, and EF2 (Voorhees
R et al., Science 330: 835-8 (2010); Shi X et al., J Mol Biol 419:
125-38 (2012); Chen K et al., PLoS One 8: e66446 (2013)). The SRL
(sarcin-ricin loop) was named for being the shared target of the
fungal ribotoxin sarcin and the plant type II RIP ricin.
[0134] The RIP Superfamily includes RIPs, fungal ribotoxins, and
bacterial ribotoxins that interfere with ribosome translocation
functions (see Table B; Brigotti M et al., Biochem J 257: 723-7
(1989)). Most RIPs, like abrin, gelonin, ricin, and saporin,
irreversibly depurinate a specific adenine in the universally
conserved sarcin/ricin loop (SRL) of the large rRNAs of ribosomes
(e.g. A4324 in animals, A3027 in fungi, and A2660 in prokaryotes).
Most fungal ribotoxins, like .alpha.-sarcin, irreversibly cleave a
specific bond in the SRL (e.g. the bond between G4325 and A4326 in
animals, G3028 and A3029 in fungi, and G2661 and A2662 in
prokaryotes) to catalytically inhibit protein synthesis by damaging
ribosomes (Martinez-Ruiz A et al., Toxicon 37: 1549-63 (1999);
Lacadena J et al., FEMS Microbiol Rev 31: 212-37 (2007); Tan Q et
al., J Biotechnol 139: 156-62 (2009)). The bacterial protein
ribotoxins Ct, DT, and PE are classified in the RIP Superfamily
because they can inhibit protein synthesis by catalytically
damaging ribosome function and induce apoptosis efficiently with
only a few toxin molecules.
[0135] RIPs are defined by one common feature, the ability to
inhibit translation in vitro by damaging the ribosome via ribosomal
RNA (rRNA)N-glycosidase activity. By 2013, over one hundred RIPs
had been described (Walsh M, Virulence 4: 774-84 (2013)). Most RIPs
depurinate a specific adenine residue in the universally conserved
sarcin/ricin loop (SRL) of the large rRNA of both eukaryotic and
prokaryotic ribosomes. The highest number of RIPs has been found in
the following families: Caryophyllaceae, Sambucaceae,
Cucurbitaceae, Euphorbiaceae, Phytolaccaceae, and Poaceae.
[0136] Members of the RIP family are categorized into at least
three classes based on their structures. Type I RIPs, e.g. gelonin,
luffins, PAP, saporins and trichosanthins, are monomeric proteins
comprising an enzymatic domain and lacking an associated targeting
domain. Type II RIPs, e.g. abrin, ricin, Shiga toxins, are
multi-subunit, heteromeric proteins with an enzymatic A subunit and
a targeting B subunit(s) typical of binary ABx toxins (Ho M, et
al., Proc Natl Acad Sci USA 106: 20276-81 (2009)). Type III RIPs,
e.g. barley JIP60 RIP and maize b-32 RIP, are synthesized as
proenzymes that require extensive proteolytic processing for
activation (Peumans W et al., FASEB J 15: 1493-1506 (2001); Mak A
et al., Nucleic Acids Res 35: 6259-67 (2007)).
[0137] Although there is low sequence homology (<50% identity)
between members of the RIP family, their catalytic domains share
conserved tertiary structures which are superimposable such that
key residues involved in the depurination of the ribosome are
identifiable (de Virgilio M et al., Toxins 2: 2699-737 (2011);
Walsh M, Virulence 4: 774-84 (2013)). For example, the catalytic
domains of ricin and Shiga toxin are superimposable using
crystallographic data despite the 18% sequence identity of their
A-chain subunits (Fraser M et al., Nat Struct Biol 1: 59-64
(1994)).
[0138] Many enzymes and polypeptide effector regions have been used
to create cytotoxic components of immunotoxins such as, e.g.,
gelonin, saporin, pokeweed antiviral protein (PAP), bryodin,
bouganin, momordin, dianthin, momorcochin, trichokirin, luffin,
restrictocin, mitogillin, alpha-sarcin, Onconase.RTM., pancreatic
ribonuclease, Bax, eosinophil-derived neurotoxin, and angiogenin.
In particular, potently cytotoxic immunotoxins have been generated
using polypeptides derived from the RIPs: ricin, gelonin, saporin,
momordin, and PAPs (Pasqualucci L et al., Haematologica 80: 546-56
(1995)).
[0139] During their respective intoxication processes, cholera
toxins, ricins, and Shiga toxins all subcellularly route to the ER
where their catalytic domains are then released and translocated to
the cytosol. These toxins may take advantage of the host cell's
unfolded protein machinery and ERAD system to signal the host cell
to export their catalytic domains into the cytosol (see Spooner R,
Lord J, Curr Top Microbiol Immunol 357: 190-40 (2012)).
[0140] The ability of a given molecule to intracellularly route to
specific sub-cellular compartments may be assayed by the skilled
worker using techniques known in the art. This includes common
techniques in the art that can localize a molecule of interest to
any one of the following sub-cellular compartments: cytosol, ER,
and lysosome.
[0141] With regard to the claimed invention, the phrase "cytosol
targeting toxin effector polypeptide" refers to a polypeptide
derived from proteins, including naturally occurring ribotoxins and
synthetic ribotoxins, which are capable of routing intracellularly
to the cytosol after cellular internalization. Commonly, cytosolic
targeting toxin effector regions are derived from naturally
occurring protein toxins or toxin-like structures which are altered
or engineered by human intervention, however, other polypeptides,
such as, e.g., computational designed polypeptides, are within the
scope of the term as used herein (see e.g. Newton D et al., Blood
97: 528-35 (2001); De Lorenzo C et al., FEBS Lett 581: 296-300
(2007); De Lorenzo C, D'Alessio G, Curr Pharm Biotechnol 9: 210-4
(2008); Menzel C et al., Blood 111: 3830-7 (2008)). Thus, cytosolic
targeting toxin effector regions may be derived from synthetic or
engineered protein constructs with increased or decreased
ribotoxicity, and/or naturally occurring proteins that have been
otherwise altered to have a non-native characteristic. The ability
of a given molecule to provide cytosol targeting toxin effector
function(s) may be assayed by the skilled worker using techniques
known in the art.
[0142] The cytosolic targeting toxin effector regions of the
present invention may be derived from ribotoxic toxin effector
polypeptides and often overlap or completely comprise a ribotoxic
toxin effector polypeptide.
2. Proteasome Delivery Effector Polypeptides Derived from Other
Polypeptide Regions or Non-Proteinaceous Materials
[0143] There are numerous proteinaceous molecules, other than
toxin-derived molecules, which have the intrinsic ability to
localize within a cell and/or direct their own intracellular
routing, to the cytosol, ER, or any other subcellular compartment
suitable for delivery to a proteasome. Any of these polypeptides
may be used directly or derivatized into proteasome delivery
effector polypeptides for use in the present invention as long as
the intrinsic subcellular localization effector function is
preserved.
[0144] For example, numerous molecules are known to be able to
escape from endosomal compartments after being endocytosed into a
cell, including numerous naturally occurring proteins and
polypeptides, via numerous mechanisms, including pore formation,
lipid bilayer fusion, and proton sponge effects (see e.g. Varkouhi
A et al., J Control Release 151: 220-8 (2010)). Non-limiting
examples of non-toxin derived molecules with endosomal escape
functions include: viral agents like hemagglutinin HA2; vertebrate
derived polypeptides and peptides like human calcitonin derived
peptides, bovine prion protein, and sweet arrow peptide; synthetic
biomimetic peptides; and polymers with endosome disrupting
abilities (see e.g. Varkouhi A et al., J Control Release 151: 220-8
(2010)). Escape from endosomal compartments, including lysosomes,
can be measured directly and quantitated using assays known in the
art, such as, e.g., using reporter assays with horseradish
peroxidase, bovine serum albumin, fluorophores like Alexa 488, and
toxin derived polypetides (see e.g. Bartz R et al., Biochem J 435:
475-87 (2011); Gilabert-Oriol, R et al., Toxins 6: 1644-66
(2014)).
[0145] Other examples are molecules which localize to specific
intracellular compartments. Most polypeptides comprising an
endoplasmic retention/retrieval signal motif (e.g. KDEL) can
localize to the ER of a eukaryotic cell from different compartments
within the cell.
[0146] The ability of a polypeptide to intracellularly route to the
cytosol, ER, and/or lysosomal compartments of a cell from the
starting position of an early endosomal compartment can be
determined by the skilled worker using assays known in the art.
Then, the proteasome delivery effector polypeptide regions of a
source polypeptide or protein, such as, e.g., a toxin, can be
mapped and isolated by the skilled worker using standard techniques
known in the art.
3. Polypeptides Engineered to Comprise One or More Heterologous,
T-Cell Epitopes and a Proteasome Delivery Effector Polypeptide
[0147] Once a proteasome delivery effector polypeptide is obtained,
it can be engineered into a T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptide of the present
invention using the methods of the present invention. Using the
methods of the present invention, one or more T-cell epitopes are
embedded, fused, or inserted into any proteasome delivery effector
polypeptide, such as, e.g., a toxin effector polypeptide which
routes to the cytosol (which may include a ribotoxic toxin effector
polypeptide), in order to create polypeptides of the present
invention, which starting from an early endosomal compartment are
capable of delivering a T-cell epitope to the proteasome for entry
into the MHC class I pathway and subsequent MHC class I
presentation.
[0148] A given molecule's ability to deliver T-cell epitopes to the
proteasome for entry into the MHC class I pathway of a cell may be
assayed by the skilled worker using the methods described herein
and/or techniques known in the art (see Examples, infra).
Similarly, a given molecule's ability to deliver a T-cell epitope
from an early endosome compartment to a proteasome may be assayed
by the skilled worker using the methods described herein and/or
techniques known in the art.
[0149] A given molecule's ability to deliver a T-cell epitope from
an early endosome compartment to a MHC class I molecule for
presentation on the surface of a cell may be assayed by the skilled
worker using the methods described herein and/or techniques known
in the art (see Examples, infra). Similarly, a given molecule's
ability to deliver a T-cell epitope from an early endosome
compartment to a MHC class I molecule may be assayed by the skilled
worker using the methods described herein and/or techniques known
in the art.
[0150] The proteasome delivery effector polypeptides modified using
the methods of the present invention are not required to be capable
of inducing or promoting cellular internalization either before or
after modification by the methods of the present invention. In
order to make cell-targeted molecules of the present invention, the
polypeptides of the present invention may be linked, using standard
techniques known in the art, with other components known to the
skilled worker in order to provide cell-targeting and/or cellular
internalization function(s) as needed.
B. Heterologous T-Cell Epitopes
[0151] The polypeptides and cell-targeted molecules of the present
invention each comprise one or more heterologous T-cell epitopes. A
T-cell epitope is a molecular structure which is comprised by an
antigen and can be represented by a peptide or linear amino acid
sequence and. A heterologous T-cell epitope is an epitope not
already present in the source polypeptide or starting proteasome
delivery effector polypeptide that is modified using a method of
the present invention in order to create a T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptide of the present
invention.
[0152] The heterologous T-cell epitope peptide may be incorporated
into the source polypeptide via numerous methods known to the
skilled worker, including, e.g., the processes of creating one or
more amino acid substitutions within the source polypeptide, fusing
one or more amino acids to the source polypeptide, inserting one or
more amino acids into the source polypeptide, linking a peptide to
the source polypeptide, and/or a combination of the aforementioned
processes. The result is a modified variant of the source
polypeptide which comprises one or more heterologous T-cell
epitopes.
[0153] Although any T-cell epitope is contemplated as being used as
a heterologous T-cell epitope of the present invention, certain
epitopes may be selected based on desirable properties. One
objective is to create CD8+ T-cell hyper-immunized polypeptides,
meaning that the heterologous T-cell epitope is highly immunogenic
and can elicit robust immune responses in vivo when displayed
complexed with a MHC class I molecule on the surface of a cell. In
certain embodiments of the polypeptides of the present invention,
the one or more heterologous T-cell epitopes are CD8+ T-cell
epitopes.
[0154] T-cell epitopes may be derived from a number of sources,
including peptide components of proteins and peptides derived from
proteins already known or shown to be capable of eliciting a
mammalian immune response. T-cell epitopes may be created or
derived from various naturally occurring proteins. T-cell epitopes
may be derived from various naturally occurring proteins foreign to
mammals, such as, e.g., proteins of microorganisms. In particular,
infectious microorganisms may contain numerous proteins with known
antigenic and/or immunogenic properties or sub-regions or epitopes.
T-cell epitopes may be derived from mutated human proteins and/or
human proteins aberrantly expressed by malignant human cells.
[0155] T-cell epitopes may be chosen or derived from a number of
source molecules already known to be capable of eliciting a
mammalian immune response, including peptides, peptide components
of proteins, and peptides derived from proteins. For example, the
proteins of intracellular pathogens with mammalian hosts are
sources for T-cell epitopes. There are numerous intracellular
pathogens, such as viruses, bacteria, fungi, and single-cell
eukaryotes, with well-studied antigenic proteins or peptides.
T-cell epitopes can be selected or identified from human viruses or
other intracellular pathogens, such as, e.g., bacteria like
mycobacterium, fungi like toxoplasmae, and protists like
trypanosomes.
[0156] For example, there are many known immunogenic viral peptide
components of viral proteins from human viruses. Numerous human
T-cell epitopes have been mapped to peptides within proteins from
influenza A viruses, such as peptides in the proteins HA
glycoproteins FE17, S139/1, C.sub.H65, C05, hemagglutin 1 (HA1),
hemagglutinin 2 (HA2), nonstructural protein 1 and 2 (NS1 and NS
2), matrix protein 1 and 2 (M1 and M2), nucleoprotein (NP),
neuraminidase (NA)), and many of these peptides have been shown to
elicit human immune responses, such as by using ex vivo assay (see
e.g. Assarsson E et al, J Virol 82: 12241-51 (2008); Alexander J et
al., Hum Immunol 71: 468-74 (2010); Wang M et al., PLoS One 5:
e10533 (2010); Wu J et al., Clin Infect Dis 51: 1184-91 (2010); Tan
P et al., Human Vaccin 7: 402-9 (2011); Grant E et al., Immunol
Cell Biol 91: 184-94 (2013); Terajima M et al., Virol J 10: 244
(2013)). Similarly, numerous human T-cell epitopes have been mapped
to peptide components of proteins from human cytomegaloviruses
(HCMV), such as peptides in the proteins pp65 (UL83), UL128-131,
immediate-early 1 (IE-1; UL123), glycoprotein B, tegument proteins,
and many of these peptides have been shown to elicit human immune
responses, such as by using ex vivo assays (Schoppel K et al., J
Infect Dis 175: 533-44 (1997); Elkington R et al, J Virol 77:
5226-40 (2003); Gibson L et al., J Immunol 172: 2256-64 (2004);
Ryckman B et al., J Virol 82: 60-70 (2008); Sacre K et al., J Virol
82: 10143-52 (2008)).
[0157] While any T-cell epitope may be used in the compositions and
methods of the present invention, certain T-cell epitopes may be
preferred based on their known and/or empirically determined
characteristics.
[0158] In many species, the MHC gene encodes multiple MHC-I
molecular variants. Because MHC class I protein polymorphisms can
affect antigen-MHC class I complex recognition by CD8+ T-cells,
heterologous T-cell epitopes may be chosen using based on knowledge
about certain MHC class I polymorphisms and/or the ability of
certain antigen-MHC class I complexes to be recognized by T-cells
of different genotypes.
[0159] There are well-defined peptide-epitopes that are known to be
immunogenic, MHC class I restricted, and/or matched with a specific
human leukocyte antigen (HLA) variant(s). For applications in
humans or involving human target cells, HLA-Class I-restricted
epitopes can be selected or identified by the skilled worker using
standard techniques known in the art. The ability of peptides to
bind to human MHC Class I molecules can be used to predict the
immunogenic potential of putative T-cell epitopes. The ability of
peptides to bind to human MHC class I molecules can be scored using
software tools. T-cell epitopes may be chosen for use as a
heterologous T-cell epitope component of the present invention
based on the peptide selectivity of the HLA variants encoded by the
alleles more prevalent in certain human populations. For example,
the human population is polymorphic for the alpha chain of MHC
class I molecules, and the variable alleles are encoded by the HLA
genes. Certain T-cell epitopes may be more efficiently presented by
a specific HLA molecule, such as, e.g., the commonly occurring HLA
variants encoded by the HLA-A allele groups HLA-A2 and HLA-A3.
[0160] When choosing T-cell epitopes for use as a heterologous
T-cell epitope component of the present invention, multiple factors
in the process of epitope selection by MHC class I molecules may be
considered that can influence epitope generation and transport to
receptive MHC class I molecules, such as, e.g., the epitope
specificity of the following factors in the target cell:
proteasome, ERAAP/ERAP1, tapasin, and TAPs can (see e.g. Akram A,
Inman R, Clin Immunol 143: 99-115 (2012)).
[0161] When choosing T-cell epitopes for use as a heterologous
T-cell epitope component of the present invention, epitope-peptides
may be selected which best match the MHC Class I molecules present
in the cell-type or cell populations to be targeted. Different MHC
class I molecules exhibit preferential binding to particular
peptide sequences, and particular peptide-MHC class I variant
complexes are specifically recognized by the TCRs of effector
T-cells. The skilled worker can use knowledge about MHC class I
molecule specificities and TCR specificities to optimize the
selection of heterologous T-cell epitopes used in the present
invention.
[0162] In addition, multiple immunogenic T-cell epitopes for MHC
class I presentation may be embedded in the same polypeptide
component(s) for use in the targeted delivery of a plurality of
T-cell epitopes simultaneously.
C. Proteasome Delivery Effector Polypeptides which Comprise One or
More Heterologous T-Cell Epitopes Embedded or Inserted to Disrupt
an Endogenous B-Cell and/or CD4+ T-Cell Epitope Region
[0163] Despite the attractiveness of using proteasome delivery
effector polypeptides as components of therapeutics, many
polypeptides are immunogenic in extracellular spaces when
administered to vertebrates. Unwanted immunogenicity in protein
therapeutics has resulted in reduced efficacy, unpredictable
pharmacokinetics, and undesirable immune responses that limit
dosages and repeat administrations. In efforts to de-immunize
therapeutics, one main challenge is silencing or disrupting
immunogenic epitopes within a polypeptide effector domain, e.g. its
cytosolic targeting domain, while retaining the desired polypeptide
effector function(s), such as, e.g., proteasome delivery. In
addition, it is a significant challenge to disrupt immune epitopes
by amino acid substitution in a polypeptide structure while
preserving its function while simultaneously adding one or more
T-cell epitopes that will not be recognized by the immune system
until after cellular internalization, processing, and cell-surface
presentation by a target cell. Solving this challenge enables the
creation of polypeptides which exhibit desired CD8+ T-cell
immunogenicity while reducing undesired B-cell and CD4+ T-cell
immunogenicity--referred to herein as "CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized" molecules or "T-cell
epitope delivering and/or B-cell/CD4+ T-cell de-immunized"
molecules.
II. The General Structure of Cell-Targeted Molecules Comprising
T-Cell Epitope Delivering, CD8+ T-Cell Hyper-Immunized Polypeptides
of the Invention
[0164] The polypeptides of the present invention may be coupled to
numerous other polypeptides, agents, and moieties to create
cell-targeted molecules, such as, e.g. cytotoxic, cell-targeted
proteins of the present invention. Cytotoxic polypeptides and
proteins may be constructed using the T-cell epitope comprising
proteasome delivering effector polypeptides of the invention and
the addition of cell-targeting components, such as, e.g., a binding
region capable of exhibiting high affinity binding to an
extracellular target biomolecule physically-coupled to the surface
of a specific cell type(s). In addition, the B-cell epitope
de-immunized polypeptides of the present invention, whether toxic
or non-toxic, may be used as components of numerous useful
molecules for administration to mammals.
A. Cell-Targeted Molecules Comprising a Proteasome Effector
Polypeptide Comprising a Heterologous T-Cell Epitope
[0165] The present invention includes cell-targeted molecules, each
comprising 1) a cell-targeting binding region and 2) a proteasome
delivering effector polypeptide of the invention which comprises a
heterologous T-cell epitope.
Cell-Targeting Moeities
[0166] Certain molecules of the present invention comprise a T-cell
hyper-immunized proteasome delivering effector polypeptide of the
present invention linked to a cell-targeting moiety comprising a
binding region capable of specifically binding an extracellular
target biomolecule. In certain embodiments, the molecules of the
present invention comprise a single polypeptide or protein such
that the T-cell hyper-immunized proteasome delivering effector
polypeptide and cell-targeting binding region are fused together to
form a continuous polypeptide chain or cell-targeting fusion
protein.
[0167] Cell-targeting moieties of the cell-targeted molecules of
the present invention comprise molecular structures, that when
linked to a polypeptide of the present invention, are each capable
of bringing the cell-targeted molecule within close proximity to
specific cells based on molecular interactions on the surfaces of
those specific cells. Cell-targeting moieties include ligand and
polypeptides which bind to cell-surface targets.
[0168] One type of cell-targeting moiety is a proteinaceous binding
region. Binding regions of the cell-targeted molecules of the
present invention comprise one or more polypeptides capable of
selectively and specifically binding an extracellular target
biomolecule. Binding regions may comprise one or more various
polypeptide moieties, such as ligands whether synthetic or
naturally occurring ligands and derivatives thereof, immunoglobulin
derived domains, synthetically engineered scaffolds as alternatives
to immunoglobulin domains, and the like. The use of proteinaceous
binding regions in cell-targeting molecules of the invention allows
for the creation of cell-targeting molecules which are
single-chain, cell-targeting proteins.
[0169] There are numerous binding regions known in the art that are
useful for targeting polypeptides to specific cell-types via their
binding characteristics, such as ligands, monoclonal antibodies,
engineered antibody derivatives, and engineered alternatives to
antibodies.
[0170] According to one specific, but non-limiting aspect, the
binding region of the cell-targeted molecule of the present
invention comprises a naturally occurring ligand or derivative
thereof that retains binding functionality to an extracellular
target biomolecule, commonly a cell surface receptor. For example,
various cytokines, growth factors, and hormones known in the art
may be used to target the cell-targeted molecules of the present
invention to the cell-surface of specific cell types expressing a
cognate cytokine receptor, growth factor receptor, or hormone
receptor. Certain non-limiting examples of ligands include
(alternative names are indicated in parentheses) B-cell activating
factors (BAFFs, APRIL), colony stimulating factors (CSFs),
epidermal growth factors (EGFs), fibroblast growth factors (FGFs),
vascular endothelial growth factors (VEGFs), insulin-like growth
factors (IGFs), interferons, interleukins (such as IL-2, IL-6, and
IL-23), nerve growth factors (NGFs), platelet derived growth
factors, transforming growth factors (TGFs), and tumor necrosis
factors (TNFs).
[0171] According to certain other embodiments, the binding region
comprises a synthetic ligand capable of binding an extracellular
target biomolecule. One non-limiting example is antagonists to
cytotoxic T-lymphocyte antigen 4 (CTLA-4).
[0172] According to one specific, but non-limiting aspect, the
binding region may comprise an immunoglobulin-type binding region.
The term "immunoglobulin-type binding region" as used herein refers
to a polypeptide region capable of binding one or more target
biomolecules, such as an antigen or epitope. Binding regions may be
functionally defined by their ability to bind to target molecules.
Immunoglobulin-type binding regions are commonly derived from
antibody or antibody-like structures; however, alternative
scaffolds from other sources are contemplated within the scope of
the term.
[0173] Immunoglobulin (Ig) proteins have a structural domain known
as an Ig domain. Ig domains range in length from about 70-110 amino
acid residues and possess a characteristic Ig-fold, in which
typically 7 to 9 antiparallel beta strands arrange into two beta
sheets which form a sandwich-like structure. The Ig fold is
stabilized by hydrophobic amino acid interactions on inner surfaces
of the sandwich and highly conserved disulfide bonds between
cysteine residues in the strands. Ig domains may be variable (IgV
or V-set), constant (IgC or C-set) or intermediate (IgI or I-set).
Some Ig domains may be associated with a complementarity
determining region (CDR) which is important for the specificity of
antibodies binding to their epitopes. Ig-like domains are also
found in non-immunoglobulin proteins and are classified on that
basis as members of the Ig superfamily of proteins. The HUGO Gene
Nomenclature Committee (HGNC) provides a list of members of the
Ig-like domain containing family.
[0174] An immunoglobulin-type binding region may be a polypeptide
sequence of an antibody or antigen-binding fragment thereof wherein
the amino acid sequence has been varied from that of a native
antibody or an Ig-like domain of a non-immunoglobulin protein, for
example by molecular engineering or selection by library screening.
Because of the relevance of recombinant DNA techniques and in vitro
library screening in the generation of immunoglobulin-type binding
regions, antibodies can be redesigned to obtain desired
characteristics, such as smaller size, cell entry, or other
therapeutic improvements. The possible variations are many and may
range from the changing of just one amino acid to the complete
redesign of, for example, a variable region. Typically, changes in
the variable region will be made in order to improve the
antigen-binding characteristics, improve variable region stability,
or reduce the potential for immunogenic responses.
[0175] There are numerous immunoglobulin-type binding regions
contemplated as components of the present invention. In certain
embodiments, the immunoglobulin-type binding region is derived from
an immunoglobulin binding region, such as an antibody paratope
capable of binding an extracellular target biomolecule. In certain
other embodiments, the immunoglobulin-type binding region comprises
an engineered polypeptide not derived from any immunoglobulin
domain but which functions like an immunoglobulin binding region by
providing high-affinity binding to an extracellular target
biomolecule. This engineered polypeptide may optionally include
polypeptide scaffolds comprising or consisting essentially of
complementary determining regions from immunoglobulins as described
herein.
[0176] There are also numerous binding regions in the prior art
that are useful for targeting polypeptides to specific cell-types
via their high-affinity binding characteristics. In certain
embodiments, the binding region of the present proteins is selected
from the group which includes single-domain antibody domains
(sdAbs), nanobodies, heavy-chain antibody domains derived from
camelids (V.sub.HH fragments), bivalent nanobodies, heavy-chain
antibody domains derived from cartilaginous fishes, immunoglobulin
new antigen receptors (IgNARs), V.sub.NAR fragments, single-chain
variable (scFv) fragments, multimerizing scFv fragments (diabodies,
triabodies, tetrabodies), bispecific tandem scFv fragments,
disulfide stabilized antibody variable (Fv) fragments, disulfide
stabilized antigen-binding (Fab) fragments consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H 1 domains, divalent F(ab')2
fragments, Fd fragments consisting of the heavy chain and C.sub.H1
domains, single chain Fv-C.sub.H3 minibodies, bispecific
minibodies, dimeric C.sub.H2 domain fragments (C.sub.H2D), Fc
antigen binding domains (Fcabs), isolated complementary determining
region 3 (CDR3) fragments, constrained framework region 3, CDR3,
framework region 4 (FR3-CDR3-FR4) polypeptides, small modular
immunopharmaceutical (SMIP) domains, and any genetically
manipulated counterparts of the foregoing that retain its paratope
and binding function (see Saerens D et al., Curr. Opin. Pharmacol
8: 600-8 (2008); Dimitrov D, MAbs 1: 26-8 (2009); Weiner L, Cell
148: 1081-4 (2012); Ahmad Z et al., Clin Dev Immunol 2012: 980250
(2012)).
[0177] In accordance with certain other embodiments, the binding
region includes engineered, alternative scaffolds to immunoglobulin
domains that exhibit similar functional characteristics, such as
high-affinity and specific binding of target biomolecules, and
enables the engineering of improved characteristics, such as
greater stability or reduced immunogenicity. For certain
embodiments of the cell-targeted proteins of the present invention,
the binding region is selected from the group which includes
engineered, fibronectin-derived, 10.sup.th fibronectin type III
(10Fn3) domain (monobodies, AdNectins.TM., or AdNexins.TM.);
engineered, tenascin-derived, tenascin type III domain
(Centryns.TM.); engineered, ankyrin repeat motif containing
polypeptide (DARPins.TM.); engineered,
low-density-lipoprotein-receptor-derived, A domain (LDLR-A)
(Avimers.TM.); lipocalin (anticalins); engineered, protease
inhibitor-derived, Kunitz domain; engineered, Protein-A-derived, Z
domain (Affibodies.TM.); engineered, gamma-B crystalline-derived
scaffold or engineered, ubiquitin-derived scaffold (Affilins);
Sac7d-derived polypeptides (Nanoffitins.RTM. or affitins);
engineered, Fyn-derived, SH2 domain (Fynomers.RTM.); miniproteins;
C-type lectin-like domain scaffolds; engineered antibody mimics;
and any genetically manipulated counterparts of the foregoing that
retains its binding functionality (Worn A, Pluckthun A, J Mol Biol
305: 989-1010 (2001); Xu L et al., Chem Biol 9: 933-42 (2002);
Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004); Binz H et
al., Nat Biotechnol 23: 1257-68 (2005); Hey T et al., Trends
Biotechnol 23:514-522 (2005); Holliger P, Hudson P, Nat Biotechnol
23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8
(2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Byla
P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules
16: 2467-85 (2011)).
[0178] Any of the above binding regions may be used as a component
of the present invention as long as the binding region component
has a dissociation constant of 10.sup.-5 to 10.sup.-12 moles per
liter, preferably less than 200 nanomolar (nM), towards an
extracellular target biomolecule.
[0179] Certain cell-targeted molecules of the present invention
comprise a polypeptide of the present invention linked to an
extracellular target biomolecule specific binding region comprising
one or more polypeptides capable of selectively and specifically
binding an extracellular target biomolecule. Extracellular target
biomolecules may be selected based on numerous criteria.
Extracellular Target Biomolecules of the Cell-Targeting
Moieties
[0180] Certain binding regions of the cell-targeted molecules of
the present invention comprise a polypeptide region capable of
binding specifically to an extracellular target biomolecule,
preferably which is physically-coupled to the surface of a cell
type of interest, such as a cancer cell, tumor cell, plasma cell,
infected cell, or host cell harboring an intracellular
pathogen.
[0181] The term "target biomolecule" refers to a biological
molecule, commonly a protein or a protein modified by
post-translational modifications, such as glycosylation, which is
capable of being bound by a binding region to target a protein to a
specific cell-type or location within an organism. Extracellular
target biomolecules may include various epitopes, including
unmodified polypeptides, polypeptides modified by the addition of
biochemical functional groups, and glycolipids (see e.g. U.S. Pat.
No. 5,091,178; EP 2431743). It is desirable that an extracellular
target biomolecule be endogenously internalized or be readily
forced to internalize upon interaction with a cell-targeted
molecule of the present invention.
[0182] For purposes of the present invention, the term
"extracellular" with regard to modifying a target biomolecule
refers to a biomolecule that has at least a portion of its
structure exposed to the extracellular environment. Extracellular
target biomolecules include cell membrane components, transmembrane
spanning proteins, cell membrane-anchored biomolecules,
cell-surface-bound biomolecules, and secreted biomolecules.
[0183] With regard to the present invention, the phrase "physically
coupled" when used to describe a target biomolecule means both
covalent and/or non-covalent intermolecular interactions that
couple the target biomolecule, or a portion thereof, to the outside
of a cell, such as a plurality of non-covalent interactions between
the target biomolecule and the cell where the energy of each single
interaction is on the order of about 1-5 kiloCalories (e.g.
electrostatic bonds, hydrogen bonds, Van der Walls interactions,
hydrophobic forces, etc.). All integral membrane proteins can be
found physically coupled to a cell membrane, as well as peripheral
membrane proteins. For example, an extracellular target biomolecule
might comprise a transmembrane spanning region, a lipid anchor, a
glycolipid anchor, and/or be non-covalently associated (e.g. via
non-specific hydrophobic interactions and/or lipid binding
interactions) with a factor comprising any one of the
foregoing.
[0184] The binding regions of the cell-targeted molecules of the
present invention may be designed or selected based on numerous
criteria, such as the cell-type specific expression of their target
biomolecules and/or the physical localization of their target
biomolecules with regard to specific cell types. For example,
certain cytotoxic proteins of the present invention comprise
binding domains capable of binding cell-surface targets which are
expressed exclusively by only one cell-type to the cell
surface.
[0185] All nucleated vertebrate cells are believed to be capable of
presenting intracelular peptide epitopes using the MHC class I
system. Thus, extracellular target biomolecules of the
cell-targeted molecules of the invention may in principle target
any nucleated vertebrate cell for T-cell epitope delivery into the
MHC class I presentation pathway.
[0186] Extracellular target biomolecules of the binding region of
the cell-targeted molecules of the present invention may include
biomarkers over-proportionately or exclusively present on cancer
cells, immune cells, and cells infected with intracellular
pathogens, such as viruses, bacteria, fungi, prions, or
protozoans.
[0187] The skilled worker, using techniques known in the art, can
link the T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptides of the present invention to various other
molecules to target specific extracellular target biomolecules
physically coupled to cells and promote target cell
internalization. For example, a polypeptide of the invention may be
linked to cell-surface receptor targeting molecule which is more
readily endocytosed, such as, e.g., via receptor mediated
endocytosis, or to a molecule which promotes cellular
internalization via mechanisms at the cell surface, such as, e.g.
promoting clathrin coated pit assembly, phospholipid layer
deformation, and/or tubular invagination. The ability of a
cell-targeting moiety to facilitate cellular internalization after
target binding may be determined using assays known to the skilled
worker.
Endoplasmic Reticulum Retention/Retrieval Signal Motif of a Member
of the KDEL Family
[0188] For purposes of the present invention, the phrase
"endoplasmic reticulum retention/retrieval signal motif," KDEL-type
signal motif, or signal motif refers to any member of the KDEL
family capable of functioning within a eukaryotic cell to promote
subcellular localization of a protein to the endoplasmic reticulum
via KDEL receptors.
[0189] The carboxy-terminal lysine-asparagine-glutamate-leucine
(KDEL) sequence is a canonical, endoplasmic reticulum retention and
retrieval signal motif for soluble proteins in eukaryotic cells and
is recognized by the KDEL receptors (see, Capitani M, Sallese M,
FEBS Lett 583: 3863-71 (2009), for review). The KDEL family of
signal motifs includes many KDEL-like motifs, such as HDEL, RDEL,
WDEL, YDEL, HEEL, KEEL, REEL, KFEL, KIEL, DKEL, KKEL, HNEL, HTEL,
KTEL, and HVEL, all of which are found at the carboxy-terminals of
proteins which are known to be residents of the lumen of the
endoplasmic reticulum of throughout multiple phylogenetic kingdoms
(Munro S, Pelham H, Cell 48: 899-907 (1987); Raykhel I et al., J
Cell Biol 179: 1193-204 (2007)). The KDEL signal motif family
includes at least 46 polypeptide variants shown using synthetic
constructs (Raykhel, J Cell Biol 179: 1193-204 (2007)). Additional
KDEL signal motifs include ALEDEL, HAEDEL, HLEDEL, KLEDEL, IRSDEL,
ERSTEL, and RPSTEL (Alanen H et al., J Mol Biol 409: 291-7 (2011)).
A generalized consensus motif representing the majority of KDEL
signal motifs has been described as [KRHQSA]-[DENQ]-E-L (Hulo N et
al., Nucleic Acids Res 34: D227-30 (2006)).
[0190] Proteins containing KDEL family signal motifs are bound by
KDEL receptors distributed throughout the Golgi complex and
transported to the endoplasmic reticulum by a microtubule-dependent
mechanism for release into the lumen of the endoplasmic reticulum
(Griffiths G et al., J Cell Biol 127: 1557-74 (1994); Miesenbock G,
Rothman J, J Cell Biol 129: 309-19 (1995)). KDEL receptors
dynamically cycle between the Golgi complex and endoplasmic
reticulum (Jackson M et al., EMBO J. 9: 3153-62 (1990); Schutze M
et al., EMBO J 13: 1696-1705 (1994)).
[0191] For purposes of the present invention, the members of the
KDEL family include synthetic signal motifs able to function within
a eukaryotic cell to promote subcellular localization of a protein
to the endoplasmic reticulum via KDEL receptors. In other words,
some members of the KDEL family might not occur in nature or have
yet to be observed in nature but have or may be constructed and
empirically verified using methods known in the art; see e.g.,
Raykhel I et al., J Cell Biol 179: 1193-204 (2007).
[0192] As a component of certain embodiments of the polypeptides
and cell-targeted molecules of the present invention, the KDEL-type
signal motif is physically located, oriented, or arranged within
the polypeptide or cell-targeted protein such that it is on a
carboxy-terminal.
[0193] For the purposes of the present invention, the specific
order or orientation is not fixed for the T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptide and the
cell-targeting binding region in relation to each other or the
entire, cell-targeted, fusion protein's N-terminal(s) and
C-terminal(s) (see e.g. FIG. 1).
[0194] The general structure of the cell-targeted molecules of the
present invention is modular, in that various, diverse
cell-targeting binding regions may be used with various CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides
to provide for diverse targeting of various extracellular target
biomolecules and thus targeting of cytotoxicity, cytostasis, and/or
exogenous material delivery to various diverse cell types. CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized
polypeptides which do not result in T-cell epitope presentation
and/or are not cytotoxic due to improper subcellular routing may
still be useful as components of cell targeted molecules for
delivering exogenous materials into cells, such as, e.g., a T-cell
epitope or antigen.
III. Linkages Connecting Polypeptide Components of the Invention
and/or their Subcomponents
[0195] Individual cell-targeting moiety, polypeptide, and/or
protein components of the present invention, e.g. the
cell-targeting binding regions and CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptides, may be
suitably linked to each other via one or more linkers well known in
the art and/or described herein. Individual polypeptide
subcomponents of the binding regions, e.g. heavy chain variable
regions (V.sub.H), light chain variable regions (V.sub.L), CDR,
and/or ABR regions, may be suitably linked to each other via one or
more linkers well known in the art and/or described herein (see
e.g. Weisser N, Hall J, Biotechnol Adv 27: 502-20 (2009); Chen X et
al., Adv Drug Deliv Rev 65: 1357-69 (2013)). Protein components of
the invention, e.g., multi-chain binding regions, may be suitably
linked to each other or other polypeptide components of the
invention via one or more linkers well known in the art. Peptide
components of the invention, e.g., KDEL family endoplasmic
reticulum retention/retrieval signal motifs, may be suitably linked
to another component of the invention via one or more linkers, such
as a proteinaceous linker, which are well known in the art.
[0196] Suitable linkers are generally those which allow each
polypeptide component of the present invention to fold with a
three-dimensional structure very similar to the polypeptide
components produced individually without any linker or other
component. Suitable linkers include single amino acids, peptides,
polypeptides, and linkers lacking any of the aforementioned such as
various non-proteinaceous carbon chains, whether branched or cyclic
(see e.g. Chen X et al., Adv Drug Deliv Rev 65: 1357-69
(2013)).
[0197] Suitable linkers may be proteinaceous and comprise one or
more amino acids, peptides, and/or polypeptides. Proteinaceous
linkers are suitable for both recombinant fusion proteins and
chemically linked conjugates. A proteinaceous linker typically has
from about 2 to about 50 amino acid residues, such as, e.g., from
about 5 to about 30 or from about 6 to about 25 amino acid
residues. The length of the linker selected will depend upon a
variety of factors, such as, e.g., the desired property or
properties for which the linker is being selected (see e.g. Chen X
et al., Adv Drug Deliv Rev 65: 1357-69 (2013)).
[0198] Suitable linkers may be non-proteinaceous, such as, e.g.
chemical linkers (see e.g. Dosio F et al., Toxins 3: 848-83 (2011);
Feld J et al., Oncotarget 4: 397-412 (2013)). Various
non-proteinaceous linkers known in the art may be used to link
cell-targeting moieties to the CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptide components, such as
linkers commonly used to conjugate immunoglobulin-derived
polypeptides to heterologous polypeptides. For example, polypeptide
regions may be linked using the functional side chains of their
amino acid residues and carbohydrate moieties such as, e.g., a
carboxy, amine, sulfhydryl, carboxylic acid, carbonyl, hydroxyl,
and/or cyclic ring group. For example, disulfide bonds and
thioether bonds may be used to link two or more polypeptides (see
e.g. Fitzgerald D et al., Bioconjugate Chem 1: 264-8 (1990);
Pasqualucci L et al., Haematologica 80: 546-56 (1995)). In
addition, non-natural amino acid residues may be used with other
functional side chains, such as ketone groups (see e.g. Sun S et
al., Chembiochem Jul. 18 (2014); Tian F et al., Proc Natl Acad Sci
USA 111: 1766-71 (2014)). Examples of non-proteinaceous chemical
linkers include but are not limited to N-succinimidyl
(4-iodoacetyl)-aminobenzoate, S--(N-succinimidyl) thioacetate
(SATA), N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio)
toluene (SMPT), N-succinimidyl 4-(2-pyridyldithio)-pentanoate
(SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate
(SMCC or MCC), sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate,
4-succinimidyl-oxycarbonyl-.alpha.-(2-pyridyldithio) toluene,
sulfosuccinimidyl-6-(.alpha.-methyl-.alpha.-(pyridyldithiol)-toluamido)
hexanoate, N-succinimidyl-3-(-2-pyridyldithio)-proprionate (SPDP),
succinimidyl 6(3(-(-2-pyridyldithio)-proprionamido) hexanoate,
sulfosuccinimidyl 6(3(-(-2-pyridyldithio)-propionamido) hexanoate,
maleimidocaproyl (MC),
maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl
(MC-vc-PAB), 3-maleimidobenzoic acid N-hydroxysuccinimide ester
(MBS), alpha-alkyl derivatives, sulfoNHS-ATMBA (sulfosuccinimidyl
N-[3-(acetylthio)-3-methylbutyryl-beta-alanine]),
sulfodicholorphenol, 2-iminothiolane, 3-(2-pyridyldithio)-propionyl
hydrazide, Ellman's reagent, dichlorotriazinic acid, and
S-(2-thiopyridyl)-L-cysteine (see e.g. Thorpe P et al., Eur J
Biochem 147: 197-206 (1985); Thorpe P et al., Cancer Res 47:
5924-31 (1987); Thorpe P et al., Cancer Res 48: 6396-403 (1988);
Grossbard M et al., Blood 79: 576-85 (1992); Lui C et al., Proc
Natl Acad Sci USA 93: 8618-23 (1996); Doronina S et al., Nat
Biotechnol 21: 778-84 (2003); Feld J et al., Oncotarget 4: 397-412
(2013)).
[0199] Suitable linkers, whether proteinaceous or
non-proteinaceous, may include, e.g., protease sensitive,
environmental redox potential sensitive, pH sensitive, acid
cleavable, photocleavable, and/or heat sensitive linkers (see e.g.
Dosio F et al., Toxins 3: 848-83 (2011); Chen X et al., Adv Drug
Deliv Rev 65: 1357-69 (2013); Feld J et al., Oncotarget 4: 397-412
(2013)).
[0200] Proteinaceous linkers may be chosen for incorporation into
recombinant fusion cell-targeted molecules of the present
invention. For recombinant fusion cell-targeted proteins of the
invention, linkers typically comprise about 2 to 50 amino acid
residues, preferably about 5 to 30 amino acid residues (Argos P, J
Mol Biol 211: 943-58 (1990); Williamson M, Biochem J 297: 240-60
(1994); George R, Heringa J, Protein Eng 15: 871-9 (2002); Kreitman
R, AAPS J 8: E532-51 (2006)). Commonly, proteinaceous linkers
comprise a majority of amino acid residues with polar, uncharged,
and/or charged residues, such as, e.g., threonine, proline,
glutamine, glycine, and alanine (see e.g. Huston J et al. Proc Natl
Acad Sci U.S.A. 85: 5879-83 (1988); Pastan I et al., Annu Rev Med
58: 221-37 (2007); Li J et al., Cell Immunol 118: 85-99 (1989);
Cumber A et al. Bioconj Chem 3: 397-401 (1992); Friedman P et al.,
Cancer Res 53: 334-9 (1993); Whitlow M et al., Protein Engineering
6: 989-95 (1993); Siegall C et al., J Immunol 152: 2377-84 (1994);
Newton et al. Biochemistry 35: 545-53 (1996); Ladurner et al. J Mol
Biol 273: 330-7 (1997); Kreitman R et al., Leuk Lymphoma 52: 82-6
(2011); U.S. Pat. No. 4,894,443). Non-limiting examples of
proteinaceous linkers include
alanine-serine-glycine-glycine-proline-glutamate (ASGGPE),
valine-methionine (VM), alanine-methionine (AM), AM(G.sub.2 to
4S).sub.xAM where G is glycine, S is serine, and x is an integer
from 1 to 10.
[0201] Proteinaceous linkers may be selected based upon the
properties desired. Proteinaceous linkers may be chosen by the
skilled worker with specific features in mind, such as to optimize
one or more of the fusion molecule's folding, stability,
expression, solubility, pharmacokinetic properties, pharmacodynamic
properties, and/or the activity of the fused domains in the context
of a fusion construct as compared to the activity of the same
domain by itself. For example, proteinaceous linkers may be
selected based on flexibility, rigidity, and/or cleavability (see
e.g. Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)). The
skilled worker may use databases and linker design software tools
when choosing linkers. Certain linkers may be chosen to optimize
expression (see e.g. Turner D et al., J Immunol Methods 205: 43-54
(1997)). Certain linkers may be chosen to promote intermolecular
interactions between identical polypeptides or proteins to form
homomultimers or different polypeptides or proteins to form
heteromultimers. For example, proteinaceous linkers may be selected
which allow for desired non-covalent interactions between
polypeptide components of the cell-targeted proteins of the
invention, such as, e.g., interactions related to the formation
dimers and other higher order multimers (see e.g. U.S. Pat. No.
4,946,778).
[0202] Flexible proteinaceous linkers are often greater than 12
amino acid residues long and rich in small, non-polar amino acid
residues, polar amino acid residues, and/or hydrophilic amino acid
residues, such as, e.g., glycines, serines, and threonines (see
e.g. Bird R et al., Science 242: 423-6 (1988); Friedman P et al.,
Cancer Res 53: 334-9 (1993); Siegall C et al., J Immunol 152:
2377-84 (1994)). Flexible proteinaceous linkers may be chosen to
increase the spatial separation between components and/or to allow
for intramolecular interactions between components. For example,
various "GS" linkers are known to the skilled worker and are
composed of multiple glycines and/or one or more serines, sometimes
in repeating units, such as, e.g., (G.sub.xS).sub.n,
(S.sub.xG).sub.n, (GGGGS).sub.n, and (G).sub.n. in which x is 1 to
6 and n is 1 to 30 (see e.g. WO 96/06641). Non-limiting examples of
flexible proteinaceous linkers include GKSSGSGSESKS,
GSTSGSGKSSEGKG, GSTSGSGKSSEGSGSTKG, GSTSGSGKPGSGEGSTKG,
EGKSSGSGSESKEF, SRSSG, and SGSSC.
[0203] Rigid proteinaceous linkers are often stiff alpha-helical
structures and rich in proline residues and/or one or more
strategically placed prolines (see Chen X et al., Adv Drug Deliv
Rev 65: 1357-69 (2013)). Rigid linkers may be chosen to prevent
intramolecular interactions between linked components.
[0204] Suitable linkers may be chosen to allow for in vivo
separation of components, such as, e.g., due to cleavage and/or
environment-specific instability (see Dosio F et al., Toxins 3:
848-83 (2011); Chen X et al., Adv Drug Deliv Rev 65: 1357-69
(2013)). In vivo cleavable proteinaceous linkers are capable of
unlinking by proteolytic processing and/or reducing environments
often at a specific site within an organism or inside a certain
cell type (see e.g. Doronina S et al., Bioconjug Chem 17: 144-24
(2006); Erickson H et al., Cancer Res 66: 4426-33 (2006)). In vivo
cleavable proteinaceous linkers often comprise protease sensitive
motifs and/or disulfide bonds formed by one or more cysteine pairs
(see e.g. Pietersz G et al., Cancer Res 48: 4469-76 (1998); The J
et al., J Immunol Methods 110: 101-9 (1998); see Chen X et al., Adv
Drug Deliv Rev 65: 1357-69 (2013)). In vivo cleavable proteinaceous
linkers may be designed to be sensitive to proteases that exist
only at certain locations in an organism, compartments within a
cell, and/or become active only under certain physiological or
pathological conditions (such as, e.g., proteases with abnormally
high levels, proteases overexpressed at certain disease sites, and
proteases specifically expressed by a pathogenic microorganism).
For example, there are proteinaceous linkers known in the art which
are cleaved by proteases present only intracellularly, proteases
present only within specific cell types, and proteases present only
under pathological conditions like cancer or inflammation, such as,
e.g., R-x-x-R motif and AMGRSGGGCAGNRVGSSLSCGGLNLQAM.
[0205] In certain embodiments of the cell-targeted molecules of the
present invention, a linker may be used which comprises one or more
protease sensitive sites to provide for cleavage by a protease
present within a target cell. In certain embodiments of the
cell-targeted molecules of the invention, a linker may be used
which is not cleavable to reduce unwanted toxicity after
administration to a vertebrate organism.
[0206] Suitable linkers may include, e.g., protease sensitive,
environmental redox potential sensitive, pH sensitive, acid
cleavable, photocleavable, and/or heat sensitive linkers, whether
proteinaceous or non-proteinaceous (see Chen X et al., Adv Drug
Deliv Rev 65: 1357-69 (2013)).
[0207] Suitable cleavable linkers may include linkers comprising
cleavable groups which are known in the art such as, e.g., Zarling
D et al., J Immunol 124: 913-20 (1980); Jung S, Moroi M, Biochem
Biophys Acta 761: 152-62 (1983); Bouizar Z et al., Eur J Biochem
155: 141-7 (1986); Park L et al., J Biol Chem 261: 205-10 (1986);
Browning J, Ribolini A, J Immunol 143: 1859-67 (1989); Joshi S,
Burrows R, J Biol Chem 265: 14518-25 (1990)).
[0208] Suitable linkers may include pH sensitive linkers. For
example, certain suitable linkers may be chosen for their
instability in lower pH environments to provide for dissociation
inside a subcellular compartment of a target cell. For example,
linkers that comprise one or more trityl groups, derivatized trityl
groups, bismaleimideothoxy propane groups, adipic acid dihydrazide
groups, and/or acid labile transferrin groups, may provide for
release of components of the cell-targeted molecules of the
invention, e.g. a polypeptide component, in environments with
specific pH ranges (see e.g. Welhoner H et al., J Biol Chem 266:
4309-14 (1991); Fattom A et al., Infect Immun 60: 584-9 (1992)).
Certain linkers may be chosen which are cleaved in pH ranges
corresponding to physiological pH differences between tissues, such
as, e.g., the pH of tumor tissue is lower than in healthy tissues
(see e.g. U.S. Pat. No. 5,612,474).
[0209] Photocleavable linkers are linkers that are cleaved upon
exposure to electromagnetic radiation of certain wavelength ranges,
such as light in the visible range (see e.g. Goldmacher V et al.,
Bioconj Chem 3: 104-7 (1992)). Photocleavable linkers may be used
to release a component of a cell-targeted molecule of the
invention, e.g. a polypeptide component, upon exposure to light of
certain wavelengths. Non-limiting examples of photocleavable
linkers include a nitrobenzyl group as a photocleavable protective
group for cysteine, nitrobenzyloxycarbonyl chloride cross-linkers,
hydroxypropylmethacrylamide copolymer, glycine copolymer,
fluorescein copolymer, and methylrhodamine copolymer (Hazum E et
al., Pept Proc Eur Pept Symp, 16th, Brunfeldt K, ed., 105-110
(1981); Senter et al., Photochem Photobiol 42: 231-7 (1985); Yen et
al., Makromol Chem 190: 69-82 (1989); Goldmacher V et al., Bioconj
Chem 3: 104-7 (1992)). Photocleavable linkers may have particular
uses in linking components to form cell-targeted molecules of the
invention designed for treating diseases, disorders, and conditions
that can be exposed to light using fiber optics.
[0210] In certain embodiments of the cell-targeted molecules of the
present invention, a cell-targeting binding region is linked to a
CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
polypeptide using any number of means known to the skilled worker,
including both covalent and noncovalent linkages (see e.g. Chen X
et al., Adv Drug Deliv Rev 65: 1357-69 (2013); Behrens C, Liu B,
MAbs 6: 46-53 (2014).
[0211] In certain embodiments of the cell-targeted proteins of the
present invention, the protein comprises a binding region which is
a scFv with a linker connecting a heavy chain variable (V.sub.H)
domain and a light chain variable (V.sub.L) domain. There are
numerous linkers known in the art suitable for this purpose, such
as, e.g., the 15-residue (Gly4Ser).sub.3 peptide. Suitable scFv
linkers which may be used in forming non-covalent multivalent
structures include GGS, GGGS, GGGGS, GGGGSGGG, GGSGGGG,
GSTSGGGSGGGSGGGGSS, and GSTSGSGKPGSSEGSTKG (Pluckthun A, Pack P,
Immunotechnology 3: 83-105 (1997); Atwell J et al., Protein Eng 12:
597-604 (1999); Wu A et al., Protein Eng 14: 1025-33 (2001); Yazaki
P et al., J Immunol Methods 253: 195-208 (2001); Carmichael J et
al., J Mol Biol 326: 341-51 (2003); Amdt M et al., FEBS Lett 578:
257-61 (2004); Bie C et al., World J Hepatol 2: 185-91 (2010)).
[0212] Suitable methods for linkage of the components of the
cell-targeted molecules of the present invention may be by any
method presently known in the art for accomplishing such, as long
as the attachment does not substantially impede the binding
capability of the cell-targeting moiety, the cellular
internalization of the CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptide component, and/or when
appropriate the desired toxin effector function(s) as measured by
an appropriate assay, including assays described herein.
[0213] For the purposes of the cell-targeted molecules of the
present invention, the specific order or orientation is not fixed
for the cell-targeting binding region and CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide
region in relation to each other or the entire cell-targeted
molecule (see e.g. FIG. 1). The components of the polypeptides and
cell-targeted molecules of the present invention may be arranged in
any order provided that the desired activities of the cell-targeted
moiety and the T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized effector polypeptide region are not eliminated. In
certain embodiments of the cell-targeted molecules of the present
invention, the cell-targeting moiety, CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptide, and/or
endoplasmic reticulum retention/retrieval signal motif may be
directly linked to each other and/or suitably linked to each other
via one or more intervening polypeptide sequences, such as with one
or more linkers well known in the art and/or described herein.
IV. Examples of Specific Structural Variations of T-Cell Epitope
Delivering, CD8+ T-Cell Hyper-Immunized Polypeptides and
Cell-Targeted Fusion Proteins Comprising the Same
[0214] A T-cell hyper-immunized polypeptide with the capability of
delivering a T-cell epitope for MHC class I presentation by a
target cell may be created, in principle, by adding a T-cell
epitope to any proteasome delivering effector polypeptide. A
B-cell/CD4+ T-cell de-immunized sub-variant of the T-cell
hyper-immunized polypeptide of the present invention may be created
by replacing one or more amino acid residues in any B-cell and/or
CD4+ T-cell epitope region within a proteasome delivering effector
polypeptide with an overlapping heterologous T-cell epitope. A
cell-targeted molecule with the ability to deliver a CD8+ T-cell
epitope for MHC class I presentation by a target cell may be
created, in principle, by linking any CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptide of the invention
to a cell-targeting moiety as long as the resulting molecule has a
cellular internalization capability provided by at least the
polypeptide of the invention, the cell-targeting moiety, or the
structural combination of them together.
[0215] A CD8+ T-cell hyper-immunized polypeptide with the
capability of delivering a CD8+ T-cell epitope for MHC class I
presentation by a target cell may be created by using a
toxin-derived, proteasome delivering effector polypeptide.
Similarly, a B-cell/CD4+ T-cell de-immunized, CD8+ T-cell
hyper-immunized polypeptide of the present invention may be created
by replacing one or more amino acid residues in any B-cell epitope
region in a toxin-derived, proteasome-delivering effector
polypeptide with an overlapping heterologous CD8+ T-cell
epitope.
[0216] Certain T-cell hyper-immunized and B-cell/CD4+ T-cell
de-immunized polypeptides of the present invention comprise a
disruption of at least one putative B-cell epitope region by the
addition of a heterologous T-cell epitope in order to reduce the
antigenic and/or immunogenic potential of the polypeptides after
administration to a mammal. The terms "disrupted" or "disruption"
or "disrupting" as used herein with regard to a B-cell epitope
region refers to the deletion of at least one amino acid in a
B-cell epitope region, inversion of two or more amino acids where
at least one of the inverted amino acids is in a B-cell epitope
region, insertion of at least one amino acid in a B-cell epitope
region, or mutation of at least one amino acid in a B-cell epitope
region. A B-cell epitope region disruption by mutation includes
amino acid substitutions with non-standard amino acids and/or
non-natural amino acids. The number of amino acid residues in the
region affected by the disruption is preferably two or more, three
or more, four or more, five or more, six or more, seven or more and
so forth up to 8, 9, 10, 11, 12, or more amino acid residues.
[0217] Certain B-cell epitope regions and disruptions are indicated
herein by reference to specific amino acid positions of native
Shiga toxin A Subunits or a prototypical Diphtheria toxin A Subunit
provided in the Sequence Listing, noting that naturally occurring
toxin A Subunits may comprise precursor forms containing signal
sequences of about 22 amino acids at their amino-terminals which
are removed to produce mature toxin A Subunits and are recognizable
to the skilled worker.
[0218] Certain T-cell hyper-immunized and B-cell/CD4+ T-cell
de-immunized polypeptides of the present invention comprise a
disruption of at least one putative CD4+ T-cell epitope region by
the addition of a heterologous T-cell epitope in order to reduce
the CD4+ T-cell antigenic and/or immunogenic potential of the
polypeptides after administration to a mammal. The terms
"disrupted" or "disruption" or "disrupting" as used herein with
regard to a CD4+ T-cell epitope region refers to the deletion of at
least one amino acid in a CD4+ T-cell epitope region, inversion of
two or more amino acids where at least one of the inverted amino
acids is in a CD4+ T-cell epitope, insertion of at least one amino
acid in a CD4+ T-cell epitope region, or mutation of at least one
amino acid in a CD4+ T-cell epitope region. A CD4+ T-cell epitope
region disruption by mutation includes amino acid substitutions
with non-standard amino acids and/or non-natural amino acids. The
number of amino acid residues in the region affected by the
disruption is preferably two or more, three or more, four or more,
five or more, six or more, seven or more and so forth up to 8, 9,
10, 11, 12, or more amino acid residues.
[0219] Certain CD4+ T-cell epitope regions and disruptions are
indicated herein by reference to specific amino acid positions of
native Shiga toxin A Subunits or a prototypical Diphtheria toxin A
Subunit provided in the Sequence Listing, noting that naturally
occurring toxin A Subunits may comprise precursor forms containing
signal sequences of about 22 amino acids at their amino-terminals
which are removed to produce mature toxin A Subunits and are
recognizable to the skilled worker.
1. Shiga Toxin Derived, CD8+ T-Cell Epitope Presenting, and
B-Cell/CD4+ T-Cell De-Immunized Polypeptides
[0220] The Shiga toxin family of protein toxins is composed of
various naturally occurring toxins which are structurally and
functionally related, e.g., Shiga toxin, Shiga-like toxin 1, and
Shiga-like toxin 2 (Johannes L, Romer W, Nat Rev Microbiol 8:
105-16 (2010)). Members of the Shiga toxin family share the same
overall structure and mechanism of action (Engedal, N et al.,
Microbial Biotech 4: 32-46 (2011)). For example, Stx, SLT-1 and
SLT-2 display indistinguishable enzymatic activity in cell free
systems (Head S et al., J Biol Chem 266: 3617-21 (1991); Tesh V et
al., Infect Immun 61: 3392-402 (1993); Brigotti M et al., Toxicon
35:1431-1437 (1997)).
[0221] The Shiga toxin family encompasses true Shiga toxin (Stx)
isolated from S. dysenteriae serotype 1, Shiga-like toxin 1
variants (SLT1 or Stx1 or SLT-1 or Slt-I) isolated from serotypes
of enterohemorrhagic E. coli, and Shiga-like toxin 2 variants (SLT2
or Stx2 or SLT-2) isolated from serotypes of enterohemorrhagic E.
coli. SLT1 differs by only one residue from Stx, and both have been
referred to as Verocytotoxins or Verotoxins (VTs) (O'Brien, Curr
Top Microbiol Immunol 180: 65-94 (1992)). Although SLT1 and SLT2
variants are only about 53-60% similar to each other at the amino
acid sequence level, they share mechanisms of enzymatic activity
and cytotoxicity common to the members of the Shiga toxin family
(Johannes, Nat Rev Microbiol 8: 105-16 (2010)). Over 39 different
Shiga toxins have been described, such as the defined subtypes
Stx1a, Stx1c, Stx1d, and Stx2a-g (Scheutz F et al., J Clin
Microbiol 50: 2951-63 (2012)). Members of the Shiga toxin family
are not naturally restricted to any bacterial species because
Shiga-toxin-encoding genes can spread among bacterial species via
horizontal gene transfer (Strauch E et al., Infect Immun 69:
7588-95 (2001); Zhaxybayeva O, Doolittle W, Curr Biol 21: R242-6
(2011)). As an example of interspecies transfer, a Shiga toxin was
discovered in a strain of A. haemolyticus isolated from a patient
(Grotiuz G et al., J Clin Microbiol 44: 3838-41 (2006)). Once a
Shiga toxin encoding polynucleotide enters a new subspecies or
species, the Shiga toxin amino acid sequence is presumed to be
capable of developing slight sequence variations due to genetic
drift and/or selective pressure while still maintaining a mechanism
of cytotoxicity common to members of the Shiga toxin family (see
Scheutz, J Clin Microbiol 50: 2951-63 (2012)).
[0222] For purposes of the present invention, the phrase "Shiga
toxin effector region" refers to a polypeptide region derived from
a Shiga toxin A Subunit of a member of the Shiga toxin family that
is capable of exhibiting at least one Shiga toxin function. Shiga
toxin functions include, e.g., cell entry, lipid membrane
deformation, directing subcellular routing, catalytically
inactivating ribosomes, effectuating cytotoxicity, and effectuating
cytostatic effects.
[0223] For purposes of the present invention, a Shiga toxin
effector function is a biological activity conferred by a
polypeptide region derived from a Shiga toxin A Subunit.
Non-limiting examples of Shiga toxin effector functions include
cellular internalization, subcellular routing, catalytic activity,
and cytotoxicity. Non-limiting examples of Shiga toxin catalytic
activities include ribosome inactivation, protein synthesis
inhibition, N-glycosidase activity, polynucleotide:adenosine
glycosidase activity, RNAase activity, and DNAase activity. RIPs
can depurinate nucleic acids, polynucleosides, polynucleotides,
rRNA, ssDNA, dsDNA, mRNA (and polyA), and viral nucleic acids
(Barbieri L et al., Biochem J 286: 1-4 (1992); Barbieri L et al.,
Nature 372: 624 (1994); Ling J et al., FEBS Lett 345: 143-6 (1994);
Barbieri L et al., Biochem J 319: 507-13 (1996); Roncuzzi L,
Gasperi-Campani A, FEBS Lett 392: 16-20 (1996); Stirpe F et al.,
FEBS Lett 382: 309-12 (1996); Barbieri L et al., Nucleic Acids Res
25: 518-22 (1997); Wang P, Turner N, Nucleic Acids Res 27: 1900-5
(1999); Barbieri L et al., Biochim Biophys Acta 1480: 258-66
(2000); Barbieri L et al., J Biochem 128: 883-9 (2000); Bagga S et
al., J Biol Chem 278: 4813-20 (2003); Picard D et al., J Biol Chem
280: 20069-75 (2005)). Some RIPs show antiviral activity and
superoxide dismutase activity (Erice A et al., Antimicrob Agents
Chemother 37: 835-8 (1993); Au T et al., FEBS Lett 471: 169-72
(2000); Parikh B, Turner N, Mini Rev Med Chem 4: 523-43 (2004);
Sharma N et al., Plant Physiol 134: 171-81 (2004)). Shiga toxin
catalytic activities have been observed both in vitro and in vivo.
Assays for Shiga toxin effector activity can measure various
activities, such as, e.g., protein synthesis inhibitory activity,
depurination activity, inhibition of cell growth, cytotoxicity,
supercoiled DNA relaxation activity, and/or nuclease activity.
[0224] As used herein, the retention of Shiga toxin effector
function refers to a level of Shiga toxin functional activity, as
measured by an appropriate quantitative assay with reproducibility
comparable to a wild-type Shiga toxin effector polypeptide control.
For ribosome inhibition, Shiga toxin effector function is
exhibiting an IC.sub.50 of 10,000 pM or less. For cytotoxicity in a
target positive cell kill assay, Shiga toxin effector function is
exhibiting a CD.sub.50 of 1,000 nM or less, depending on the cell
type and its expression of the appropriate extracellular target
biomolecule.
[0225] As used herein, the retention of "significant" Shiga toxin
effector function refers to a level of Shiga toxin functional
activity, as measured by an appropriate quantitative assay with
reproducibility comparable to a wild-type Shiga toxin effector
polypeptide control. For in vitro ribosome inhibition, significant
Shiga toxin effector function is exhibiting an IC.sub.50 of 300 pM
or less depending on the source of the ribosomes (e.g. bacteria,
archaea, or eukaryote (algae, fungi, plants, or animals)). This is
significantly greater inhibition as compared to the approximate
IC.sub.50 of 100,000 pM for the catalytically inactive SLT-1A 1-251
double mutant (Y77S, E167D). For cytotoxicity in a target positive
cell kill assay in laboratory cell culture, significant Shiga toxin
effector function is exhibiting a CD.sub.50 of 100, 50, or 30 nM or
less, depending on the cell line and its expression of the
appropriate extracellular target biomolecule. This is significantly
greater cytotoxicity to the appropriate target cell line as
compared to the SLT-1A component alone, without a cell-targeting
binding region, which has a CD.sub.50 of 100-10,000 nM, depending
on the cell line.
[0226] For some samples, accurate values for either IC.sub.50 or
CD.sub.50 might be unobtainable due to the inability to collect the
required data points for an accurate curve fit. Inaccurate
IC.sub.50 and/or CD.sub.50 values should not be considered when
determining significant Shiga toxin effector function activity.
Data insufficient to accurately fit a curve as described in the
analysis of the data from exemplary Shiga toxin effector function
assays, such as, e.g., assays described in the Examples, should not
be considered as representative of actual Shiga toxin effector
function. For example, theoretically neither an IC.sub.50 nor
CD.sub.50 can be determined if greater than 50% ribosome inhibition
or cell death, respectively, does not occur in a concentration
series for a given sample.
[0227] The failure to detect activity in Shiga toxin effector
function may be due to improper expression, polypeptide folding,
and/or polypeptide stability rather than a lack of cell entry,
subcellular routing, and/or enzymatic activity. Assays for Shiga
toxin effector functions may not require much polypeptide of the
invention to measure significant amounts of Shiga toxin effector
function activity. To the extent that an underlying cause of low or
no effector function is determined empirically to relate to protein
expression or stability, one of skill in the art may be able to
compensate for such factors using protein chemistry and molecular
engineering techniques known in the art, such that a Shiga toxin
functional effector activity may be restored and measured. As
examples, improper cell based expression may be compensated for by
using different expression control sequences; improper polypeptide
folding and/or stability may benefit from stabilizing terminal
sequences, or compensatory mutations in non-effector regions which
stabilize the three dimensional structure of the protein, etc. When
new assays for individual Shiga toxin functions become available,
CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
Shiga toxin effector polypeptides may be analyzed for any level of
those Shiga toxin effector functions, such as a being within
1000-fold or 100-fold or less the activity of a wild-type Shiga
toxin effector polypeptide or exhibiting 3-fold to 30-fold or
greater activity as compared to a functional knockout Shiga toxin
effector polypeptide.
[0228] Sufficient subcellular routing may be merely deduced by
observing cytotoxicity in cytotoxicity assays, such as, e.g.,
cytotoxicity assays based on T-cell epitope presentation or based
on a toxin effector function involving a cytosolic and/or ER target
substrate.
[0229] It should be noted that even if the cytotoxicity of a Shiga
toxin effector polypeptide is reduced relative to wild-type, in
practice, applications using attenuated CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized Shiga toxin effector
polypeptides may be equally or more effective than those using
wild-type Shiga toxin effector polypeptides because the reduced
antigenicity and/or immunogenicity might offset the reduced
cytotoxicity, such as, e.g., by allowing higher dosages, more
repeated administrations, or chronic administration. Wild-type
Shiga toxin effector polypeptides are very potent, being able to
kill with only one molecule reaching the cytosol or perhaps 40
molecules being internalized. CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized Shiga toxin effector polypeptides
with even considerably reduced Shiga toxin effector functions, such
as, e.g., subcellular routing or cytotoxicity, as compared to
wild-type Shiga toxin effector polypeptides may still be potent
enough for applications based on targeted cell killing and/or
specific cell detection.
[0230] Certain embodiments of the present invention provide
polypeptides comprising a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of a
member of the Shiga toxin Family, the Shiga toxin effector region
comprising a disruption of at least one natively positioned B-cell
epitope region provided herein (see e.g. Tables 2, 3, and 4). In
certain embodiments, a CD8+ T-cell hyper-immunized and B-cell/CD4+
T-cell de-immunized Shiga toxin effector polypeptide of the
invention may comprise or consist essentially of full-length Shiga
toxin A Subunit (e.g. SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), or
SLT-2A (SEQ ID NO:3)) comprising at least one disruption of the
amino acid sequence selected from the group of natively positioned
amino acids consisting of: the B-cell epitope regions 1-15 of SEQ
ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3;
27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ
ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2,
or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID
NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID
NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID
NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and
210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID
NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278
of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1
or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQ ID
NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2, 77-103
of SEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID
NO:2, 160-183 of SEQ ID NO: 1 or SEQ ID NO:2, 236-258 of SEQ ID NO:
1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; or
the equivalent position in a conserved Shiga toxin effector
polypeptide and/or non-native Shiga toxin effector polypeptide
sequence.
[0231] Certain embodiments of the present invention provide
polypeptides comprising a Shiga toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of a
member of the Shiga toxin Family, the Shiga toxin effector region
comprising a disruption of at least one natively positioned CD4+
T-cell epitope region provided herein (see e.g. Tables 2, 3, and
4). In certain embodiments, a CD8+ T-cell hyper-immunized and
B-cell/CD4+ T-cell de-immunized Shiga toxin effector polypeptide of
the invention may comprise or consist essentially of full-length
Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO: 1), StxA (SEQ ID
NO:2), or SLT-2A (SEQ ID NO:3)) comprising at least one disruption
of the amino acid sequence selected from the group of natively
positioned amino acids consisting of: 4-33, 34-78, 77-103, 128-168,
160-183, 236-258, and 274-293; or the equivalent position in a
conserved Shiga toxin effector polypeptide and/or non-native Shiga
toxin effector polypeptide sequence.
[0232] In certain embodiments, a Shiga toxin effector polypeptide
of the present invention may comprise or consist essentially of a
truncated Shiga toxin A Subunit. Truncations of Shiga toxin A
Subunits might result in the deletion of entire B-cell epitope
regions without affecting toxin effector catalytic activity and
cytotoxicity. The smallest Shiga toxin A Subunit fragment
exhibiting significant enzymatic activity is a polypeptide composed
of residues 75-247 of StxA (Al-Jaufy, Infect Immun 62: 956-60
(1994)). Truncating the carboxy-terminus of SLT-1A, StxA, or SLT-2A
to amino acids 1-251 removes two predicted B-cell epitope regions,
two predicted CD4 positive (CD4+) T-cell epitopes, and a predicted
discontinuous B-cell epitope. Truncating the amino-terminus of
SLT-1A, StxA, or SLT-2A to 75-293 removes at least three predicted
B-cell epitope regions and three predicted CD4+ T-cell epitopes.
Truncating both amino- and carboxy-terminals of SLT-1A, StxA, or
SLT-2A to 75-251 deletes at least five predicted B-cell epitope
regions, four putative CD4+ T-cell epitopes, and one predicted
discontinuous B-cell epitope.
[0233] In certain embodiments, a Shiga toxin effector polypeptide
of the present invention may comprise or consist essentially of a
full-length or truncated Shiga toxin A Subunit with at least one
mutation, e.g. deletion, insertion, inversion, or substitution, in
a provided B-cell and/or CD4+ T-cell epitope region. In certain
further embodiments, the polypeptides comprise a disruption which
comprises a deletion of at least one amino acid within the B-cell
and/or CD4+ T-cell epitope region. In certain further embodiments,
the polypeptides comprise a disruption which comprises an insertion
of at least one amino acid within the B-cell and/or CD4+ T-cell
epitope region. In certain further embodiments, the polypeptides
comprise a disruption which comprises an inversion of amino acids,
wherein at least one inverted amino acid is within the B-cell
and/or CD4+ T-cell epitope region. In certain further embodiments,
the polypeptides comprise a disruption which comprises a mutation,
such as an amino acid substitution to a non-standard amino acid or
an amino acid with a chemically modified side chain. Numerous
examples of amino acid substitutions are provided in the
Examples.
[0234] In other embodiments, the Shiga toxin effector polypeptides
of the present invention comprises or consists essentially of a
truncated Shiga toxin A Subunit which is shorter than a full-length
Shiga toxin A Subunit wherein at least one amino acid is disrupted
in a natively positioned B-cell and/or CD4+ T-cell epitope region
provided in the Examples (see Tables 2, 3, and/or 4).
[0235] The CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized Shiga toxin effector polypeptides of the invention may
be smaller than the full length A subunit, such as, e.g.,
consisting of the polypeptide region from amino acid position 77 to
239 (SLT-1A (SEQ ID NO: 1) or StxA (SEQ ID NO:2)) or the equivalent
in other A Subunits of members of the Shiga toxin family (e.g. 77
to 238 of (SEQ ID NO:3)). For example, in certain embodiments of
the present invention, the Shiga toxin effector polypeptides
derived from SLT-1A may be derived from amino acids 75 to 251 of
SEQ ID NO:1, 1 to 241 of SEQ ID NO:1, 1 to 251 of SEQ ID NO:1, or
amino acids 1 to 261 of SEQ ID NO:1 wherein at least one amino acid
is disrupted in an endogenous B-cell and/or CD4+ T-cell epitope
region provided in the Examples (Tables 2, 3, and/or 4). Similarly,
CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
Shiga toxin effector regions derived from StxA may comprise or
consist essentially of amino acids 75 to 251 of SEQ ID NO:2, 1 to
241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or amino acids 1 to
261 of SEQ ID NO:2 wherein at least one amino acid is disrupted in
at least one endogenous B-cell and/or CD4+ T-cell epitope region
provided in the Examples (Tables 2, 3, and/or 4). Additionally, the
Shiga toxin effector regions derived from SLT-2 may comprise or
consist essentially of amino acids 75 to 251 of SEQ ID NO:3, 1 to
241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3, or amino acids 1 to
261 of SEQ ID NO:3 wherein at least one amino acid is disrupted in
at least one B-cell and/or CD4+ T-cell epitope region provided in
the Examples (Tables 2, 3, and/or 4).
[0236] Certain embodiments of the cell-targeted molecules of the
present invention each comprise a CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized Shiga toxin effector
polypeptide which retains a Shiga toxin effector function but which
may be engineered from a cytotoxic parental molecule to a
polypeptide with diminished or abolished cytotoxicity for
non-cytotoxic functions, e.g., effectuating cytostasis, delivery of
exogenous materials, and/or detection of cell types, by mutating
one or more key residues for enzymatic activity.
[0237] For certain embodiments, the polypeptides of the present
invention comprise Shiga toxin effector polypeptides. For certain
embodiments, the polypeptides of the present invention comprise or
consist essentially of one of the polypeptides of SEQ ID NOs:
11-43.
[0238] For certain embodiments, the cell-targeted molecules of the
present invention are cytotoxic proteins comprising Shiga toxin
effector polypeptides. For certain embodiments, the cell-targeted
molecules of the present invention comprise or consist essentially
of one of the polypeptides of SEQ ID NOs: 49-54.
2. Diphtheria Toxin Derived, CD8+ T-Cell Hyper-Immunized and/or
B-Cell/CD4+ T-Cell De-Immunized Polypeptides
[0239] For purposes of the present invention, the phrase
"diphtheria toxin effector region" refers to a polypeptide region
derived from a diphtheria toxin of a member of the Diphtheria toxin
family that is capable of exhibiting at least one diphtheria toxin
function. Diphtheria toxin functions include, e.g., cell entry,
endosome escape, directing subcellular routing, catalytically
inactivating ribosomes, effectuating cytotoxicity, and effectuating
cytostatic effects.
[0240] For purposes of the present invention, a diphtheria toxin
effector function is a biological activity conferred by a
polypeptide region derived from a diphtheria toxin. Non-limiting
examples of diphtheria toxin effector functions include cellular
internalization, subcellular routing, catalytic activity, and
cytotoxicity. Non-limiting examples of diphtheria toxin catalytic
activities include ribosome inactivation, protein synthesis
inhibition, and ADP-ribosylation. Diphtheria toxin catalytic
activities have been observed both in vitro and in vivo. Assays for
diphtheria toxin effector activity can measure various activities,
such as, e.g., protein synthesis inhibitory activity,
ADP-ribosylation, inhibition of cell growth, and/or cytotoxicity.
Sufficient subcellular routing may be merely deduced by observing
cytotoxicity in cytotoxicity assays, such as, e.g., cytotoxicity
assays based on T-cell epitope presentation or based on a toxin
effector function involving a cytosolic and/or ER target
substrate.
[0241] It should be noted that even if a toxin effector activity of
a diphtheria toxin effector polypeptide is reduced relative to
wild-type, in practice, applications using attenuated CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized diphtheria
toxin effector polypeptides may be equally or more effective than
those using diphtheria toxin effector polypeptides with wild-type
levels of activity because the reduced antigenicity and/or
immunogenicity might offset the reduced cytotoxicity, such as,
e.g., by allowing higher dosages, more repeated administrations, or
chronic administration. Diphtheria toxin effector polypeptides
exhibiting only the effector activity of subcellular routing are
appropriate for use in applications based on targeted cell CD8+
T-cell epitope delivery.
[0242] Certain embodiments of the present invention provide
polypeptides comprising a diphtheria toxin effector polypeptide
comprising an amino acid sequence derived from an A Subunit of a
member of the Diphtheria toxin Family, the diphtheria toxin
effector region comprising a disruption of at least one natively
positioned B-cell and/or CD4+ T-cell epitope region provided herein
(see e.g. Table 5). In certain embodiments, a CD8+ T-cell
hyper-immunized and B-cell/CD4+ T-cell de-immunized diphtheria
toxin effector polypeptide of the invention may comprise or consist
essentially of the polypeptide of amino acids 2-389 of SEQ ID NO:45
comprising at least one disruption of the amino acid sequence
selected from the group of natively positioned amino acids
consisting of: 3-10 of SEQ ID NO:44, 15-31 of SEQ ID NO:44, 32-54
of SEQ ID NO:44; 33-43 of SEQ ID NO:44, 71-77 of SEQ ID NO:44,
93-113 of SEQ ID NO:44, 125-131 of SEQ ID NO:44, 138-146 of SEQ ID
NO:44, 141-167 of SEQ ID NO:44, 165-175 of SEQ ID NO:44, 182-201 of
SEQ ID NO:45, 185-191 of SEQ ID NO:44, and 225-238 of SEQ ID NO:45;
or the equivalent position in a conserved diphtheria toxin effector
polypeptide and/or non-native diphtheria toxin effector polypeptide
sequence.
[0243] Optionally, the diphtheria toxin effector polypeptide of the
invention may comprise one or more mutations (e.g. substitutions,
deletions, insertions or inversions) as compared to wild-type as
long as at least one amino acid is disrupted in at least one
natively positioned B-cell and/or CD4+ T-cell epitope region
provided in the Examples (see Table 5). In certain embodiments of
the invention, the CD8+ T-cell hyper-immunized and/or B-cell/CD4+
T-cell de-immunized diphtheria toxin effector polypeptides have
sufficient sequence identity to a naturally occurring diphtheria
toxin A Subunit to retain cytotoxicity after entry into a cell,
either by well-known methods of host cell transformation,
transfection, infection or induction, or by internalization
mediated by a cell-targeting binding region linked with the
diphtheria toxin effector polypeptide.
[0244] The most critical residues for enzymatic activity and/or
cytotoxicity in the diphtheria toxin A Subunits have been mapped to
the following residue-positions: histidine-21, tyrosine-27,
glycine-52, tryptophan-50, tyrosine-54, tyrosine-65, glutamate-148,
and tryptophan-153 (Tweten R et al., J Biol Chem 260: 10392-4
(1985); Wilson B et al., J Biol Chem 269: 23296-301 (1994); Bell C,
Eisenberg D, Biochemistry 36: 481-8 (1997); Cummings M et al.,
Proteins 31: 282-98 (1998); Keyvani K et al., Life Sci 64: 1719-24
(1999); Dolan K et al., Biochemistry 39: 8266-75 (2000);
Zdanovskaia M et al., Res Micrbiol 151: 557-62 (2000); Kahn K,
Bruice T, J Am Chem Soc 123: 11960-9 (2001); Malito E et al., Proc
Natl Acad Sci USA 109: 5229-34 (2012)). The capacity of a
cytotoxic, cell-targeted molecule of the invention to cause cell
death, e.g. its cytotoxicity, may be measured using any one or more
of a number of assays well known in the art.
[0245] Among certain embodiments of the present invention, the
polypeptides comprise the CD8+ T-cell hyper-immunized and
B-cell/CD4+ T-cell de-immunized diphteria toxin effector comprising
or consisting essentially of amino acids 2 or amino acids 2-389 of
SEQ ID NO:45 wherein at least one amino acid is disrupted in the
natively positioned B-cell epitope and/or CD4+ T-cell epitope
regions provided in the Examples (Table 5).
[0246] For certain embodiments, the polypeptides of the present
invention comprise diphtheria toxin effector polypeptides. For
certain embodiments, the polypeptides of the present invention
comprise or consist essentially of one of the polypeptides of SEQ
ID NOs: 46-48.
[0247] For certain embodiments, the cell-targeted molecules of the
present invention are cytotoxic proteins comprising diphtheria
toxin effector polypeptides. For certain embodiments, the
cell-targeted molecules of the present invention comprise or
consist essentially of one of the polypeptides of SEQ ID NOs:
55-60.
[0248] For certain embodiments, the polypeptide of the present
invention comprises or consists essentially of any one of the
polypeptides of SEQ ID NOs: 11-43 or 46-48.
[0249] Cell-targeted molecules of the present invention each
comprise at least one T-cell hyper-immunized and/or B-cell/CD4+
T-cell de-immunized polypeptide linked to a cell-targeting moiety
which can bind specifically to at least one extracellular target
biomolecule in physical association with a cell, such as a target
biomolecule expressed on the surface of a cell. This general
structure is modular in that any number of diverse cell-targeting
moieties may be linked to the CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptides of the present
invention.
[0250] It is within the scope of the invention to use fragments,
variants, and/or derivatives of the polypeptides and cell-targeted
molecules of the present invention which contain a functional
binding site to any extracellular part of a target biomolecule, and
even more preferably capable of binding a target biomolecule with
high affinity (e.g. as shown by K.sub.D). Any cell-targeting moiety
which binds an extracellular part of a target biomolecule with a
dissociation constant (K.sub.D) of 10.sup.-5 to 10.sup.-12
moles/liter, preferably less than 200 nM, may be substituted for
use in making cell-targeted molecules of the invention and methods
of the invention.
VI. Variations in the Polypeptide Sequence of the T-Cell
Hyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized Polypeptides
of the Invention and Cell-Targeted Molecules Comprising the
Same
[0251] The skilled worker will recognize that variations may be
made to T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptides and cell-targeted molecules of the
present invention, and polynucleotides encoding any of the former,
without diminishing their biological activities, e.g., by
maintaining the overall structure and function of the toxin
effector region in conjunction with one or more epitope disruptions
which reduce antigenic and/or immunogenic potential. For example,
some modifications may facilitate expression, purification, and/or
pharmacokinetic properties, and/or immunogenicity. Such
modifications are well known to the skilled worker and include, for
example, a methionine added at the amino terminus to provide an
initiation site, additional amino acids placed on either terminus
to create conveniently located restriction sites or termination
codons, and biochemical affinity tags fused to either terminus to
provide for convenient detection and/or purification.
[0252] Also contemplated herein is the inclusion of additional
amino acid residues at the amino and/or carboxy termini, such as
sequences for epitope tags or other moieties. The additional amino
acid residues may be used for various purposes including, e.g.,
facilitating cloning, facilitating expression, post-translational
modification, facilitating synthesis, purification, facilitating
detection, and administration. Non-limiting examples of epitope
tags and moieties are chitin binding protein domains,
enteropeptidase cleavage sites, Factor Xa cleavage sites, FIAsH
tags, FLAG tags, green fluorescent proteins (GFP),
glutathione-S-transferase moieties, HA tags, maltose binding
protein domains, myc tags, polyhistidine tags, ReAsH tags,
strep-tags, strep-tag II, TEV protease sites, thioredoxin domains,
thrombin cleavage site, and V5 epitope tags.
[0253] In certain of the above embodiments, the polypeptide
sequence of the CD8+ T-cell hyper-immunized and/or B-cell/CD4+
T-cell de-immunized polypeptides and/or cell-targeted proteins of
the invention are varied by one or more conservative amino acid
substitutions introduced into the polypeptide region(s) as long as
at least one amino acid is disrupted in at least one natively
positioned B-cell epitope region provided herein. As used herein,
the term "conservative substitution" denotes that one or more amino
acids are replaced by another, biologically similar amino acid
residue. Examples include substitution of amino acid residues with
similar characteristics, e.g. small amino acids, acidic amino
acids, polar amino acids, basic amino acids, hydrophobic amino
acids and aromatic amino acids (see, for example, Table C, infra).
An example of a conservative substitution with a residue normally
not found in endogenous, mammalian peptides and proteins is the
conservative substitution of an arginine or lysine residue with,
for example, ornithine, canavanine, aminoethylcysteine, or another
basic amino acid. For further information concerning phenotypically
silent substitutions in peptides and proteins see, e.g., Bowie J et
al., Science 247: 1306-10 (1990).
TABLE-US-00003 TABLE C Examples of Conservative Amino Acid
Substitutions I II III IV V VI VII VIII IX X XI XII XIII XIV A D H
C F N A C F A C A A D G E K I W Q G M H C D C C E P Q R L Y S I P W
F E D D G S N M T L Y G H G E K T V V H K N G P I N P H Q L Q S K R
M R T N S R S V Q T T T R V S W P Y T
[0254] In the conservative substitution scheme in Table C,
exemplary conservative substitutions of amino acids are grouped by
physicochemical properties--I: neutral, hydrophilic; II: acids and
amides; III: basic; IV: hydrophobic; V: aromatic, bulky amino
acids, VI hydrophilic uncharged, VII aliphatic uncharged, VIII
non-polar uncharged, IX cycloalkenyl-associated, X hydrophobic, XI
polar, XII small, XIII turn-permitting, and XIV flexible. For
example, conservative amino acid substitutions include the
following: 1) S may be substituted for C; 2) M or L may be
substituted for F; 3) Y may be substituted for M; 4) Q or E may be
substituted for K; 5) N or Q may be substituted for H; and 6) H may
be substituted for N.
[0255] Additional conservative amino acid substitutions include the
following: 1) S may be substituted for C; 2) M or L may be
substituted for F; 3) Y may be substituted for M; 4) Q or E may be
substituted for K; 5) N or Q may be substituted for H; and 6) H may
be substituted for N.
[0256] In certain embodiments, the CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptides and/or
cell-targeted molecules (e.g. cell-targeted proteins) of the
present invention may comprise functional fragments or variants of
a polypeptide region of the invention that have, at most, 20, 15,
10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.
[0257] In certain embodiments, the CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptides and/or
cell-targeted molecules of the present invention may comprise
functional fragments or variants of a polypeptide region of the
invention that have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1 amino acid substitutions compared to a polypeptide sequence
recited herein, as long as it retains a disruption of at least one
amino acid in a natively positioned B-cell and/or CD4+ T-cell
epitope region provided in the Examples (Tables 2, 3, 4, and/or 5)
and as long as the polypeptides or proteins retain a T-cell epitope
delivery functionality alone and/or as a component of a therapeutic
and/or diagnostic composition. Variants of the CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga toxin
effector polypeptides and/or cell-targeted proteins of the
invention are within the scope of the invention as a result of
changing a polypeptide of the cell-targeted protein of the
invention by altering one or more amino acids or deleting or
inserting one or more amino acids, such as within the binding
region or the CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptide region, in order to achieve desired
properties, such as changed cytotoxicity, changed cytostatic
effects, changed immunogenicity, and/or changed serum half-life. A
B-cell epitope de-immunized and CD8+ T-cell hyper-immunized
polypeptide and/or a cell-targeted protein of the invention may
further be with or without a signal sequence.
[0258] Accordingly, in certain embodiments, the Shiga toxin
effector or diphtheria toxin effector polypeptides of the present
invention comprise or consists essentially of amino acid sequences
having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, 99%, 99.5% or 99.7% overall sequence identity to a naturally
occurring toxin, such as, e.g., Shiga toxin A Subunit, such as
SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID
NO:3), or a diphtheria toxin catalytic domain (SEQ ID NO: 44), in
certain embodiments, the de-immunized Shiga toxin effector or
diphtheria toxin effector polypeptides of the present invention
comprise or consists essentially of amino acid sequences having at
least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,
99.5% or 99.7% overall sequence identity to a naturally occurring
toxin wherein at least one amino acid is disrupted in at least one
natively positioned B-cell and/or CD4+ T-cell epitope region
provided in the Examples (Tables 2, 3, 4, and/or 5).
[0259] In certain embodiments of the cell-targeted molecules of the
present invention, one or more amino acid residues may be mutated,
inserted, or deleted in order to increase the enzymatic activity of
the CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized toxin effector polypeptide region. For example,
mutating residue-position alanine-231 in Stx1A to glutamate
increased its enzymatic activity in vitro (Suhan M, Hovde C, Infect
Immun 66: 5252-9 (1998)).
[0260] In certain embodiments of the cell-targeted molecules of the
present invention, one or more amino acid residues may be mutated
or deleted in order to reduce or eliminate catalytic and/or
cytotoxic activity of the CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized toxin effector polypeptide region.
For example, the catalytic and/or cytotoxic activity of the A
Subunits of members of the Shiga toxin family or Diphtheria toxin
family may be diminished or eliminated by mutation or
truncation.
[0261] In certain embodiments of the present invention, the
ribotoxin effector region has been altered such that it no longer
supports catalytic inactivation of a ribosome in vitro. However,
other means of modifying a ribotoxic effector region to reduce or
eliminate ribotoxicity are also envisioned within the scope of the
present invention. For example, certain mutations can render a
ribotoxic effector region unable to bind its ribosome substrate
despite maintaining catalytic ability observable by an in vitro
assay whereas other mutations can render a ribotoxic region unable
to target a specific ribonucleic acid sequence within in the
ribosome despite maintaining catalytic ability towards naked
nucleic acids in vitro (see e.g. Alford S et al., BMC Biochem 10: 9
(2009); Alvarez-Garcia E et al., Biochim Biophys Act 1814: 1377-82
(2011); Wong Y et al., PLoS One 7: e49608 (2012)).
[0262] In DT, there are several amino acid residues known to be
important for catalytic activity, such as, e.g., histidine-21,
tyrosine-27, glycine-52, tryptophan-50, tyrosine-54, tyrosine-65,
glutamate-148, and tryptophan-153 (Tweten R et al., J Biol Chem
260: 10392-4 (1985); Wilson B et al., J Biol Chem 269: 23296-301
(1994); Bell C, Eisenberg D, Biochemistry 36: 481-8 (1997);
Cummings M et al., Proteins 31: 282-98 (1998); Keyvani K et al.,
Life Sci 64: 1719-24 (1999); Dolan K et al., Biochemistry 39:
8266-75 (2000); Zdanovskaia M et al., Res Micrbiol 151: 557-62
(2000); Kahn K, Bruice T, J Am Chem Soc 123: 11960-9 (2001); Malito
E et al., Proc Natl Acad Sci USA 109: 5229-34 (2012)).
Glutamate-581 in cholix toxin is conserved with glutamate-148 in DT
(Jorgensen R et al., EMBO Rep 9: 802-9 (2008)), and thus, mutations
of glutamate-581 in cholix toxin are predicted to reduce the
enzymatic activity of cholix toxin.
[0263] In PE, there are several amino acid residues known to be
important for catalytic activity, such as, e.g., tryptophan-417,
histidine-426, histidine-440, glycine-441, arginine-485,
tryptophan-458, tryptophan-466, tyrosine-470, tyrosine-481,
glutamate-546, arginine-551, glutamate-553, and tryptophan-558
(Douglas C, Collier R, J Bacteriol 169: 4967-71 (1987); Wilson B,
Colliver R, Curr Top Microbiol Immunol 175: 27-41 (1992)); Beattie
B et al., Biochemistry 35: 15134-42 (1996); Roberts T, Merrill A,
Biochem J 367: 601-8 (2002); Yates S et al., Biochem J 385: 667-75
(2005); Jorgensen R et al., EMBO Rep 9: 802-9 (2008)).
Glutamate-574 and glutamate-581 in cholix toxin is conserved with
glutamate-546 and glutamate-553 in PE respectively (Jorgensen R et
al., EMBO Rep 9: 802-9 (2008)), and thus, mutations of
glutamate-574 and/or glutamate-581 in cholix toxin are predicted to
reduce the enzymatic activity of cholix toxin.
[0264] Because the catalytic domains of cholix toxin, DT, PE, and
other related enzymes are superimposable (Jorgensen R, et al., J
Biol Chem 283: 10671-8 (2008)), amino acid residues required for
catalytic activity may be predicted in related polypeptide
sequences by sequence alignment methods known to the skilled
worker.
[0265] Several members of the RIP family have been well studied
with regard to catalytic residues. For example, most RIP family
members share five key amino acid residues for catalysis, such as
e.g., two tyrosines near the amino terminus of the catalytic
domain, a glutamate and arginine near the center of the catalytic
domain, and a tryptophan near the carboxy terminus of the catalytic
domain (Lebeda F, Olson M, Int J Biol Macromol 24: 19-26 (1999);
Mlsna D et al., Protein Sci 2: 429-35 (1993); de Virgilio M et al.,
Toxins 2: 2699-737 (2011); Walsh M, Virulence 4: 774-84 (2013))).
Because the catalytic domains of members of the RIP family are
superimposable, amino acid residues required for catalytic activity
may be predicted in unstudied and/or new members of the RIP family
by sequence alignment methods known to the skilled worker (see e.g.
Husain J et al., FEBS Lett 342: 154-8 (1994); Ren J et al.,
Structure 2: 7-16 (1994); Lebeda F, Olson M, Int J Biol Macromol
24: 19-26 (1999); Ma Q et al., Acta Crystallogr D Biol Crystallogr
56: 185-6 (2000); Savino C et al., FEBS Lett 470: 239-43 (2000);
Robertus J, Monzingo A, Mini Rev Med Chem 4: 477-86 (2004); Mishra
V et al., J Biol Chem 280: 20712-21 (2005); Zhou C et al.,
Bioinformatics 21: 3089-96 (2005); Lubelli C et al., Anal Biochem
355: 102-9 (2006); Touloupakis E et al., FEBS J 273: 2684-92
(2006); Hou X et al., BMC Struct Biol 7: 29 (2007); Meyer A et al.,
Biochem Biophys Res Commun 364: 195-200 (2007); Ruggiero A et al.,
Protein Pept Lett 14: 97-100 (2007); Wang T et al., Amino Acids 34:
239-43 (2008)).
[0266] In the A Subunit of abrin, there are several amino acid
residues important for catalytic activity, such as, e.g.,
tyrosine-74, tyrosine-113, glutamate-164, arginine-167, and
tryptophan-198 (Hung C et al., Eur J Biochem 219: 83-7 (1994); Chen
J et al., Protein Eng 10: 827-33 (1997); Xie L et al., Eur J
Biochem 268: 5723-33 (2001)).
[0267] In charybdin, there are several amino acid residues
important for catalytic activity, such as, e.g., valine-79,
tyrosine-117, glutamate-167, and arginine-170 (Touloupakis E et
al., FEBS J 273: 2684-92 (2006)).
[0268] In the A Subunit of cinnamon, there are several amino acid
residues important for catalytic activity, such as, e.g.,
tyrosine-75, tyrosine-115, glutamate-167, arginine-170, and
tryptophan-201 (Hung C et al., Eur J Biochem 219: 83-7 (1994); Chen
J et al., Protein Eng 10: 827-33 (1997)).
[0269] In luffaculin, there are several amino acid residues
important for catalytic activity, such as, e.g., tyrosine-70,
glutamate-85, tyrosine-110, glutamate-159, and arginine-162 (Hou X
et al., BMC Struct Biol 7: 29 (2007)).
[0270] In luffins, there are several amino acid residues important
for catalytic activity, such as, e.g., tyrosine-71, glutamate-86,
tyrosine-111, glutamate-160, and arginine-163 (Ma Q et al., Acta
Crystallogr D Biol Crystallogr 56: 185-6 (2000))
[0271] In maize RIPs, there are several amino acid residues
important for catalytic activity, such as, e.g., tyrosine-79,
tyrosine-115, glutamate-167, arginine-170, and tryptophan-201
(Robertus J, Monzingo A, Mini Rev Med Chem 4: 477-86 (2004); Yang Y
et al., J Mol Biol 395: 897-907 (2009)).
[0272] In the PD-Ls, there are several amino acid residues
important for catalytic activity, such as, e.g., tyrosine-72,
tyrosine-122, glutamate-175, arginine-178, and tryptophan-207 in
PDL-1 (Ruggiero A et al., Biopolymers 91: 1135-42 (2009)).
[0273] In the A Subunit of the mistletoe RIP, there are several
amino acid residues important for catalytic activity, such as,
e.g., tyrosine-66, phenylalanine-75, tyrosine-110, glutamate-159,
arginine-162, glutamate-166, arginine-169, and tryptophan-193
(Langer M et al., Biochem Biophys Res Commun 264: 944-8 (1999);
Mishra V et al., Act Crystallogr D Biol Crystallogr 60: 2295-2304
(2004); Mishra V et al., J Biol Chem 280: 20712-21 (2005); Wacker R
et al., J Pept Sci 11: 289-302 (2005)).
[0274] In pokeweed antiviral protein (PAP), there are several amino
acid residues important for catalytic activity, such as, e.g.,
lysine-48, tyrosine-49, arginine-67, arginine-68, asparagine-69,
asparagine-70, tyrosine-72, phenylalanine-90, asparagine-91,
aspartate-92, arginine-122, tyrosine-123, glutamate-176,
arginine-179, tryptophan-208, and lysine-210 (Rajamohan F et al., J
Biol Chem 275: 3382-90 (2000); Rajamohan F et al., Biochemistry 40:
9104-14 (2001)).
[0275] In the A chain of ricin, there are several amino acid
residues known to be important for catalytic activity, such as,
e.g., arginine-48, tyrosine-80, asparagine-122, tyrosine-123,
glutamate-177, arginine-180, serine-203, asparagine-209,
tryptophan-211, glycine-212, arginine-213, serine-215, and
isoleucine-252 (Frankel A et al., Mol Cell Biol 9: 415-20 (1989);
Schlossman D et al., Mol Cell Biol 9: 5012-21 (1989); Gould J et
al., Mol Gen Genet 230: 91-90 (1991); Ready M et al., Proteins 10:
270-8 (1991); Rutenber E et al., Proteins 10: 240-50 (1991);
Monzingo A, Robertus, J, J Mol Biol 227: 1136-45 (1992); Day P et
al., Biochemistry 35: 11098-103 (1996); Marsden C et al., Eur J
Biochem 27: 153-62 (2004); Pang Y et al., PLoS One 6: e17883
(2011)). In ricin, there are several amino acid residues which when
deleted are known to impair the catalytic activity of ricin such
as, e.g., N24, F25, A28, V29, Y81, V82, V83, G84, E146, E147, A148,
I149, S168, F169, I170, I171, C172, I173, Q174, M175, I176, S177,
E178, A179, A180, R181, F182, Q183, Y184, D202, P203, I206, T207,
N210, S211, W212, and G213 (Munishkin A, Wool I, J Biol Chem 270:
30581-7 (1995); Berrondo M, Gray J, Proteins 79: 2844-60
(2011)).
[0276] In saporins, there are several amino acid residues known to
be important for catalytic activity, such as, e.g., tyrosine-16,
tyrosine-72, tyrosine-120, glutamate-176, arginine-179, and
tryptophan-208 (Bagga S et al., J Biol Chem 278: 4813-20 (2003);
Zarovni N et al., Canc Gene Ther 14: 165-73 (2007); Lombardi A et
al., FASEB J 24: 253-65 (2010)). In addition, a signal peptide may
be included to reduce catalytic activity (Marshall R et al., Plant
J 65: 218-29 (2011)).
[0277] In trichosanthins, there are several amino acid residues
known to be important for catalytic activity, such as, e.g.,
tyrosine-70, tyrosine-111, glutamate-160, arginine-163, lysine-173,
arginine-174, lysine-177, and tryptophan-192 (Wong et al., Eur J
Biochem 221: 787-91 (1994); Li et al., Protein Eng 12: 999-1004
(1999); Yan et al., Toxicon 37: 961-72 (1999); Ding et al., Protein
Eng 16: 351-6 (2003); Guo Q et al., Protein Eng 16: 391-6 (2003);
Chan D et al., Nucleic Acid Res 35: 1660-72 (2007)).
[0278] Fungal ribotoxins enzymatically target the same universally
conserved SRL ribosomal structure as members of the RIP family and
most fungal ribotoxins share an RNase Ti type catalytic domain
sequence and secondary structure (Lacadena J et al., FEMS Microbiol
Rev 31: 212-37 (2007)). Most fungal ribotoxins and related enzymes
share three highly conserved amino acid residues for catalysis, two
histidine residues and a glutamate residue (e.g. histidine-40,
glutamate-58, and histidine-92 in RNase Ti). A DSKKP motif is often
present in fungal ribotoxins to specifically bind the SRL (Kao R,
Davies J, J Biol Chem 274: 12576-82 (1999)). Because fungal
ribotoxin catalytic domains are superimposable, amino acid residues
required for catalytic activity may be predicted in unstudied
and/or new fungal ribotoxins using one or more sequence alignment
methods known to the skilled worker.
[0279] For Aspfl, an internal deletion of 16 amino acid residues
(positions 7-22) severely impaired its ribonucleolytic activity and
cytotoxicity (Garcia-Ortega L et al., FEBS J 272: 2536-44
(2005)).
[0280] In mitogillin, there are several amino acid residues known
to be important for catalytic activity, such as, e.g.,
asparagine-7, histidine-49, glutamate-95, lysine-111, arginine-120,
and histidine-136 (Kao R et al., Mol Microbiol 29: 1019-27 (1998);
Kao R, Davies J, FEBS Lett 466: 87-90 (2000)).
[0281] In restrictocin, there are several amino acid residues known
to be important for catalytic activity, such as, e.g., tyrosine-47,
histidine-49, glutamate-95, lysine-110, lysine-111, lysine-113,
arginine-120, and histidine-136 (Nayak S, Batra J, Biochemistry 36:
13693-9 (1997); Nayak S et al., Biochemistry 40: 9115-24 (2001);
Plantinga M et al., Biochemistry 50: 3004-13 (2011)).
[0282] In .alpha.-sarcin, there are several amino acid residues
known to be important for catalytic activity, such as, e.g.,
tryptophan-48, histidine-49, histidine-50, tryptophan-51,
asparagine-54, isoleucine-69, glutamate-95, glutamate-96,
lysine-11, lysine-112, lysine-114, arginine-121, histidine-136,
histidine-137, lysine-145 (Lacadena J et al., Biochem J 309: 581-6
(1995); Lacadena J et al., Proteins 37: 474-84 (1999);
Martinez-Ruiz A et al., Toxicon 37: 1549-63 (1999); de Antonio C et
al., Proteins 41: 350-61 (2000); Masip M et al., Eur J Biochem 268:
6190-6 (2001)).
[0283] The cytotoxicity of the A Subunits of members of the Shiga
toxin family may be altered, reduced, or eliminated by mutation or
truncation. The positions labeled tyrosine-77, glutamate-167,
arginine-170, tyrosine-114, and tryptophan-203 have been shown to
be important for the catalytic activity of Stx, Stx1, and Stx2
(Hovde C et al., Proc Natl Acad Sci USA 85: 2568-72 (1988);
Deresiewicz R et al., Biochemistry 31: 3272-80 (1992); Deresiewicz
R et al., Mol Gen Genet 241: 467-73 (1993); Ohmura M et al., Microb
Pathog 15: 169-76 (1993); Cao C et al., Microbiol Immunol 38: 441-7
(1994); Suhan M, Hovde C, Infect Immun 66: 5252-9 (1998)). Mutating
both glutamate-167 and arginine-170 eliminated the enzymatic
activity of Slt-I A1 in a cell-free ribosome inactivation assay
(LaPointe, J Biol Chem 280: 23310-18 (2005)). In another approach
using de novo expression of Slt-I A1 in the endoplasmic reticulum,
mutating both glutamate-167 and arginine-170 eliminated Slt-I A1
fragment cytotoxicity at that expression level (LaPointe, J Biol
Chem 280: 23310-18 (2005)). A truncation analysis demonstrated that
a fragment of StxA from residues 75 to 268 still retains
significant enzymatic activity in vitro (Haddad, J Bacteriol 175:
4970-8 (1993)). A truncated fragment of Slt-I A1 containing
residues 1-239 displayed significant enzymatic activity in vitro
and cytotoxicity by de novo expression in the cytosol (LaPointe, J
Biol Chem 280: 23310-18 (2005)). Expression of a Slt-I A1 fragment
truncated to residues 1-239 in the endoplasmic reticulum was not
cytotoxic because it could not retrotranslocate to the cytosol
(LaPointe, J Biol Chem 280: 23310-18 (2005)).
[0284] The most critical residues for enzymatic activity and/or
cytotoxicity in the Shiga toxin A Subunits were mapped to the
following residue-positions: aspargine-75, tyrosine-77,
tyrosine-114, glutamate-167, arginine-170, arginine-176, and
tryptophan-203 among others (Di, Toxicon 57: 535-39 (2011)). In
particular, a double-mutant construct of Stx2A containing
glutamate-E167-to-lysine and arginine-176-to-lysine mutations was
completely inactivated; whereas, many single mutations in Stx1 and
Stx2 showed a 10-fold reduction in cytotoxicity. Further,
truncation of Stx1A to 1-239 or 1-240 reduced its cytotoxicity, and
similarly, truncation of Stx2A to a conserved hydrophobic residue
reduced its cytotoxicity. The most critical residues for binding
eukaryotic ribosomes and/or eukaryotic ribosome inhibition in the
Shiga toxin A Subunit have been mapped to the following
residue-positions arginine-172, arginine-176, arginine-179,
arginine-188, tyrosine-189, valine-191, and leucine-233 among
others (McCluskey A et al., PLoS One 7: e31191 (2012).
[0285] Shiga-like toxin 1 A Subunit truncations are catalytically
active, capable of enzymatically inactivating ribosomes in vitro,
and cytotoxic when expressed within a cell (LaPointe, J Biol Chem
280: 23310-18 (2005)). The smallest Shiga toxin A Subunit fragment
exhibiting full enzymatic activity is a polypeptide composed of
residues 1-239 of Slt1A (LaPointe, J Biol Chem 280: 23310-18
(2005)). Although the smallest fragment of the Shiga toxin A
Subunit reported to retain substantial catalytic activity was
residues 75-247 of StxA (Al-Jaufy, Infect Immun 62: 956-60 (1994)),
a StxA truncation expressed de novo within a eukaryotic cell
requires only up to residue 240 to reach the cytosol and exert
catalytic inactivation of ribosomes (LaPointe, J Biol Chem 280:
23310-18 (2005)).
[0286] In certain embodiments of the CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized Shiga toxin effector
polypeptides and/or cell-targeted molecules of the present
invention derived from SLT-1A (SEQ ID NO: 1) or StxA (SEQ ID NO:2),
these changes include substitution of the asparagine at position
75, tyrosine at position 77, tyrosine at position 114, glutamate at
position 167, arginine at position 170, arginine at position 176,
and/or substitution of the tryptophan at position 203. Examples of
such substitutions will be known to the skilled worker based on the
prior art, such as asparagine at position 75 to alanine, tyrosine
at position 77 to serine, substitution of the tyrosine at position
114 to serine, substitution of the glutamate position 167 to
glutamate, substitution of the arginine at position 170 to alanine,
substitution of the arginine at position 176 to lysine, and/or
substitution of the tryptophan at position 203 to alanine. Other
mutations which either enhance or reduce Shiga toxin enzymatic
activity and/or cytotoxicity are within the scope of the invention
and may be determined using well known techniques and assays
disclosed herein.
[0287] The CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptides and/or cell-targeted molecules of the
invention may optionally be conjugated to one or more additional
agents, which may include therapeutic and/or diagnostic agents
known in the art, including such agents as described herein.
V. General Functions of the CD8+ T-Cell Hyper-Immunized and/or
B-Cell/CD4+ T-Cell De-Immunized Polypeptides of the Present
Invention and Cell-Targeted Molecules Comprising the Same
[0288] The present invention describes various CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides
which may be used as components of various compositions of matter,
such as cell-targeted cytotoxic molecules and diagnostic
compositions. In particular, CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptides have uses as
components of various protein therapeutics, such as, e.g.
immunotoxins and ligand-toxin fusions, for the targeted killing of
specific cell types for the treatment of a variety of diseases,
including cancers, immune disorders, and microbial infections.
[0289] Any CD8+ T-cell hyper-immunized, polypeptide of the
invention may be engineered into a potentially useful, therapeutic,
cell-targeted molecule with the addition of a cell-targeting moiety
which targets cellular internalization to a specific cell-type(s)
within a chordate, such as, e.g., an amphibian, bird, fish, mammal,
reptile, or shark. Similarly, any B-cell epitope de-immunized
polypeptide of the invention may be engineered into a potentially
useful, therapeutic, cell-targeted molecule with the addition of a
cell-targeting moiety which targets cellular internalization to a
specific cell-type(s) within a chordate. The present invention
provides various cytotoxic cell-targeted molecules comprising CD8+
T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
polypeptides functionally associated with binding regions to
effectuate cell targeting such that the cytotoxic cell-targeted
molecules selectively delivery T-cell epitopes, kill, inhibit the
growth of, deliver exogenous material to, and/or detect specific
cell types. This system is modular, in that any number of diverse
binding regions may be used to target to diverse cell types any
CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
polypeptide of the invention, including.
[0290] The presentation of a T-cell immunogenic epitope peptide by
the MHC class I complex targets the presenting cell for killing by
CTL-mediated cytolysis. By engineering MHC class I peptides into
proteasome delivering effector polypeptide components of
target-cell-internalizing therapeutics, the targeted delivery and
presentation of immuno-stimulatory antigens may be accomplished by
harnessing vertebrate target cells' endogenous MHC class I
pathways. The presentation by targeted cells of immuno-stimulatory
non-self antigens, such as, e.g., known viral epitope-peptides with
high immunogenicity, can signal to other immune cells to destroy
the target cells and recruit more immune cells to the target cell
site within an organism.
[0291] Thus, already cytotoxic molecules, such as e.g. potential
therapeutics comprising cytotoxic toxin effector regions, may be
engineered using methods of the invention into more cytotoxic
molecules and/or to have an additional cytotoxicity mechanism
operating via effector T-cells. These multiple cytotoxic mechanisms
may complement each other (such as by providing both direct target
cell killing and indirect (CTL-mediated) cell killing, redundantly
backup each other (such as by providing one mechanism of cell
killing in the absence of the other), and/or protect against the
development of therapeutic resistance (by limiting resistance to
the less probable situation of the malignant or infected cell
evolving to block two different cell-killing mechanisms
simultaneously).
[0292] In addition, parental cytotoxic molecules which rely on
toxin and/or enzymatic regions for cytotoxicity may be engineered
to be cytotoxic only via T-cell epitope cytosolic delivery and
presentation by both embedding a T-cell epitope and inactivating
the enzymatic activity of the parental molecule, either with the
embedded T-cell epitope or independently by other means such as
mutation or truncation. This approach removes one cytotoxic
mechanism while adding another and adds the capability of
immuno-stimulation to the local area. Furthermore, parental
cytotoxic molecules which rely on toxin and/or enzymatic regions
for cytotoxicity may be engineered to be cytotoxic only via T-cell
epitope cytosolic delivery and presentation by embedding a T-cell
epitope in the enzymatic domain of the parental molecule such that
the enzymatic activity is reduced or eliminated by the sequence
changes that create the heterologous T-cell epitope. This allows
for the one-step modification of enzymatically-cytotoxic molecules,
which have the ability to internalize into cells and route to the
cytosol, into enzymatically inactive, cytotoxic molecules which
rely on T-cell epitope proteasome delivery and presentation for
cytotoxicity and local immuno-stimulation.
A. Delivery of T-Cell Epitopes for MHC Class I Presentation on a
Cell Surface
[0293] One function of certain CD8+ T-cell hyper-immunized
polypeptides and cell-targeted molecules of the present invention
is the delivery of one or more T-cell epitopes for MHC class I
presentation by a cell. Delivery of exogenous T-cell epitope
peptides to the MHC class I system of a target cell can be used to
induce the target cell to present the T-cell epitope peptide in
association with MHC class I molecules on the cell surface, which
subsequently leads to the activation of CD8+ effector T-cells to
attack the target cell.
[0294] Certain embodiments of the CD8+ T-cell hyper-immunized
polypeptides and cell-targeted molecules of the present invention
are capable of delivering one or more T-cell epitopes to the
proteasome of a target cell. The delivered T-cell epitope are then
proteolytic processed and presented by the MHC class I pathway on
the outside surface of the target cell.
[0295] The applications of these T-cell epitope presenting
functions of the CD8+ T-cell hyper-immunized polypeptides and
cell-targeted molecules of the present invention are vast. Every
nucleated cell in a mammalian organism may be capable of MHC class
I pathway presentation of immunogenic T-cell epitope peptides on
their cell outer surfaces complexed to MHC class I molecules. In
addition, the sensitivity of T-cell epitope recognition is so
exquisite that only a few MHC-I peptide complexes are required to
be presented--even presentation of a single complex can be
sufficient for recognition by an effector T-cell (Sykulev Y et al.,
Immunity 4: 565-71 (1996)).
[0296] In order for a heterologous T-cell epitope to be presented
on a target cell surface, the polypeptide delivering the
heterologous T-cell epitope-peptide must be degraded by a
proteasome in the target cell such that a peptide fragment
comprising the T-cell epitope is created and transported to the
lumen of the ER for loading onto a MHC class I molecule.
[0297] In addition, the CD8+ T-cell hyper-immunized polypeptide
must first reach the interior of a target cell and then come in
contact with a proteasome in the target cell. In order to deliver a
CD8+ T-cell hyper-immunized polypeptide of the present invention to
the interior of a target cell, cell-targeting molecules of the
present invention must be capable of target cell internalization.
Once the CD8+ T-cell hyper-immunized polypeptide of the invention
is internalized as a component of a cell-targeting molecule, the
CD8+ T-cell hyper-immunized polypeptide will typical reside in an
early endosomal compartment, such as, e.g., endocytotic vesicle.
The CD8+ T-cell hyper-immunized polypeptide then has to reach a
target cell's proteasome with at least one intact, heterologous
T-cell epitope.
[0298] These functions can be detected and monitored by a variety
of standard methods known in the art to the skilled worker. For
example, the ability of cell-targeted molecules of the present
invention to deliver a T-cell epitope peptide and drive
presentation of the epitope peptide by the MHC class I system of
target cells may be investigated using various in vitro and in vivo
assays, including, e.g., the direct detection/visualization of MHC
class I/peptide complexes, measurement of binding affinities for
the heterologous T-cell epitope peptide to MHC Class I molecules,
and/or measurement of functional consequences of MHC class
I-epitope peptide complex presentation on target cells by
monitoring CTL responses.
[0299] Certain assays to monitor this function of the polypeptides
and molecules of the present invention involve the direct detection
of a specific MHC Class I/peptide antigen complex in vitro or ex
vivo. Common methods for direct visualization and quantitation of
peptide-MHC class I complexes involve various immuno-detection
reagents known to the skilled worker. For example, specific
monoclonal antibodies can be developed to recognize a particular
MHC/Class I/peptide antigen complex (Porgador A et al, Immunity 6:
715-26 (1997)). Similarly, soluble, multimeric T cell receptors,
such as the TCR-STAR reagents (Altor, Mirmar, Fla., U.S.) can be
used to directly visualize or quantitate specific MHC I/antigen
complexes (Zhu X et al., J Immunol 176: 3223-32 (2006)). These
specific mAbs or soluble, multimeric T-cell receptors may be used
with various detection methods, including, e.g.
immunohistochemistry, flow cytometry, and enzyme-linked immuno
assay (ELISA).
[0300] An alternative method for direct identification and
quantification of MHC I/peptide complexes involves mass
spectrometry analyses, such as, e.g., the ProPresent Antigen
Presentation Assay (ProImmune, Inc., Sarasota, Fla., U.S.) in which
peptide-MCH class I complexes are extracted from the surfaces of
cells, then the peptides are purified and identified by sequencing
mass spectrometry (Falk K et al., Nature 351: 290-6 (1991)).
[0301] Certain assays to monitor the T-cell epitope delivery and
MHC class I presentation function of the polypeptides and molecules
of the present invention involve computational and/or experimental
methods to monitor MHC Class I and peptide binding and stability.
Several software programs are available for use by the skilled
worker for predicting the binding responses of epitope peptides to
MHC Class I alleles, such as, e.g., The Immune Epitope Database and
Analysis Resource (IEDB) Analysis Resource MHC-I binding prediction
Consensus tool (Kim Y et al., Nucleic Acid Res 40: W525-30 (2012).
Several experimental assays have been routinely applied, such as,
e.g. cell surface binding assays and/or surface plasmon resonance
assays to quantify and/or compare binding kinetics (Miles K et al.,
Mol Immunol 48: 728-32 (2011)). Additionally, other MHC-peptide
binding assays based on a measure of the ability of a peptide to
stabilize the ternary MHC-peptide complex for a given MHC Class I
allele, as a comparison to known controls, have been developed
(e.g., MHC-peptide binding assay from ProImmmune, Inc.).
[0302] Alternatively, measurements of the consequence of MHC Class
I/peptide antigen complex presentation on the cell surface can be
performed by monitoring the cytotoxic T cell (CTL) response to the
specific complex. These measurements by include direct labeling of
the CTLs with MHC Class I tetramer or pentamer reagents. Tetramers
or pentamers bind directly to T cell receptors of a particular
specificity, determined by the Major Histocompatibility Complex
(MHC) allele and peptide combination. Additionally, the
quantification of released cytokines, such as interferon gamma or
interleukins by ELISA or enzyme-linked immunospot (ELIspot) is
commonly assayed to identify specific CTL responses. The cytotoxic
capacity of CTL can be measured using a number of assays, including
the classical 51 Chromium (Cr) release assay or alternative
non-radioactive cytotoxicity assays (e.g., CytoTox96.RTM.
non-radioactive kits and CellTox.TM. CellTiter-GLO.RTM. kits
available from Promega Corp., Madison, Wis., U.S.), Granzyme B
ELISpot, Caspase Assays or LAMP-1 translocation flow cytometric
assays. To specifically monitor the killing of target cells,
Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used
to easily and quickly label a cell population of interest for in
vitro or in vivo investigation to monitor killing of epitope
specific CSFE labeled target cells (Durward M et al., J Vis Exp 45
pii 2250 (2010)).
[0303] In vivo responses to MHC Class I presentation can be
followed by administering a MHC Class I/antigen promoting agent
(e.g., a peptide, protein or inactivated/attenuated virus vaccine)
followed by challenge with an active agent (e.g. a virus) and
monitoring responses to that agent, typically in comparison with
unvaccinated controls. Ex vivo samples can be monitored for CTL
activity with methods similar to those described previously (e.g.
CTL cytotoxicity assays and quantification of cytokine
release).
[0304] HLA-A, HLA-B, and/or HLA-C molecules are isolated from the
intoxicated cells after lysis using immune affinity (e.g., an
anti-MHC antibody "pulldown" purification) and the associated
peptides (i.e., the peptides presented by the isolated MHC
molecules) are recovered from the purified complexes. The recovered
peptides are analyzed by sequencing mass spectrometry. The mass
spectrometry data is compared against a protein database library
consisting of the sequence of the exogenous (non-self) peptide
(T-cell epitope X) and the international protein index for humans
(representing "self" or non-immunogenic peptides). The peptides are
ranked by significance according to a probability database. All
detected antigenic (non-self) peptide sequences are listed. The
data is verified by searching against a scrambled decoy database to
reduce false hits (see e.g. Ma B, Johnson R, Mol Cell Proteomics
11: O111.014902 (2012)). The results will demonstrate that peptides
from the T-cell epitope X are presented in MHC complexes on the
surface of intoxicated target cells.
[0305] The set of presented peptide-antigen-MHC complexes can vary
between cells due to the antigen-specific HLA molecules expressed.
T-cells can then recognize specific peptide-antigen-MHC complexes
displayed on a cell surface using different TCR molecules with
different antigen-specificities.
[0306] Because multiple T-cell epitopes may be delivered by a
cell-targeted molecule of the invention, such as, e.g., by
embedding two or more different T-cell epitopes in a single
proteasome delivering effector polypeptide, a single cell-targeted
molecule of the invention may be effective chordates of the same
species with different MHC class variants, such as, e.g., in humans
with different HLA alleles. This may allow for the simultaneously
combining different T-cell epitopes with different effectiveness in
different sub-populations of subjects based on MHC complex protein
diversity and polymorphisms (see e.g. Yuhki N et al., J Hered 98:
390-9 (2007)). For example, human MHC complex proteins, HLA
proteins, vary among humans based on genetic ancestry, e.g. African
(sub-Saharan), Amerindian, Caucasiod, Mongoloid, New Guinean and
Australian, or Pacific islander (see e.g. Wang M, Claesson M,
Methods Mol Biol 1184: 309-17 (2014)).
[0307] The activation of T-cell responses are desired
characteristics of certain anti-cancer, anti-neoplastic,
anti-tumor, and/or anti-microbial biologic drugs to stimulate the
patient's own immune system toward targeted cells. Activation of a
robust and strong T-cell response is also a desired characteristic
of many vaccines (Aly H A, J Immunol Methods 382: 1-23 (2012)). The
presentation of a T-cell epitope by a target cell within an
organism can lead to the activation of robust immune responses to a
target cell and/or its general locale within an organism. Thus, the
targeted delivery of a T-cell epitope for presentation may be
utilized for engineering the activation of T-cell responses during
a therapeutic regime.
B. Cell Kill Via Targeted Cytotoxicity and/or Recruitment of
CTLs
[0308] Cell-targeted molecules of the present invention comprising
CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
polypeptides of the present invention can provide both: 1) cell
type specific T-cell-epitope delivery for MHC class I presentation
and 2) potent cytotoxicity. In addition, certain embodiments of the
cell-targeted molecules of the present invention also provide
de-immunization, which reduces the likelihood of certain immune
responses when administered to a mammal.
[0309] In certain embodiments of the cell-targeted molecules of the
present invention, upon contacting a cell physically coupled with
an extracellular target biomolecule of the cell-targeting moiety
(e.g. a cell-targeted binding region), the cell-targeted molecule
of the invention is capable of causing death of the cell. The
mechanism of cell kill may be direct, e.g. via the enzymatic
activity of a toxin effector region, or indirect via CTL-mediated
cytolysis, and may be under varied conditions of target cells, such
as an ex vivo manipulated target cell, a target cell cultured in
vitro, a target cell within a tissue sample cultured in vitro, or a
target cell in vivo.
1. Indirect Cell Kill Via T-Cell Epitope Delivery and MHC Class I
Presentation
[0310] T-cell epitope delivering, CD8+ T-cell hyper-immunized
polypeptides of the present invention, with or without B-cell
epitope de-immunization, may be used as components of cell-targeted
molecules for indirect cell kill. Certain embodiments of the
cell-targeted molecules of the present invention are cytotoxic
because they comprise a CD8+ T-cell epitope presenting polypeptide
of the invention which delivers one or more T-cell epitopes to the
MHC class I presentation pathway of a target cell upon target
internalization of the cell-targeted molecule.
[0311] In certain embodiments of the cell-targeted molecules of the
present invention, upon contacting a cell physically coupled with
an extracellular target biomolecule of the cell-targeting moiety
(e.g. a cell-targeted binding region), the cell-targeted molecule
of the invention is capable of indirectly causing the death of the
cell, such as, e.g., via the presentation of one or more T-cell
epitopes by the target cell and the subsequent recruitment of
CTLs.
2. Direct Cell Kill Via Cell-Targeted Toxin Cytotoxicity
[0312] T-cell epitope delivering, CD8+ T-cell hyper-immunized,
and/or B-cell/CD4+ T-cell de-immunized polypeptides of the present
invention may be used as components of cell-targeted molecules for
direct cell kill.
[0313] Because many naturally occurring toxins are adapted to
killing eukaryotic cells, cytotoxic proteins designed using
toxin-derived, proteasome delivering effector regions, can show
potent cell-kill activity. In particular, proteasome delivering
effector regions may also comprise ribotoxic toxin effector
polypeptides. However, other toxin effector regions are
contemplated for use in the cell-targeted molecules of the
invention, such as, e.g., polypeptides from toxins which do not
catalytically inactive ribosomes but rather are cytotoxic due to
other mechanisms. For example, cholix toxins, heat-labile
enterotoxins, and pertussis toxins heterotrimeric G proteins by
attacking the Gsalpha subunit.
[0314] The A Subunits of many members of the ABx toxin superfamily
comprise enzymatic domains capable of killing a eukaryotic cell
once in the cell's cytosol. The replacement of a B-cell epitope
with a T-cell epitope within multiple ABx toxin-derived,
polypeptides comprising toxin enzymatic domains did not
significantly alter their enzymatic activity. Thus, the CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides
of the present invention can potentially provide two mechanisms of
cell kill.
[0315] Certain embodiments of the cell-targeted molecules of the
present invention are cytotoxic because they comprise a CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide
of the invention which comprises an active toxin component.
[0316] In certain embodiments of the cell-targeted molecules of the
present invention, upon contacting a cell physically coupled with
an extracellular target biomolecule of the cell-targeting moiety
(e.g. a cell-targeted binding region), the cell-targeted molecule
of the invention is capable of directly causing the death of the
cell, such as, e.g., via the enzymatic activity of a toxin effector
region.
C. De-Immunization Improves Applications Involving Administration
to Mammals
[0317] The polypeptides and cell-targeted molecules of the present
invention have improved usefulness for administration to mammalian
species as either a therapeutic and/or diagnostic agent because of
the reduced likelihood of producing undesired immune responses in
mammals while increasing the likelihood of producing desirable
immune responses in mammals.
[0318] Certain CD8+ T-cell hyper-immunized and/or B-cell/CD4+
T-cell de-immunized toxin-derived polypeptides of the present
invention might differ in their antigenicity profiles when
administered to various mammals, but are expected to have reduced
B-cell and/or CD4+ T-cell antigenicity and/or immunogenicity. In
certain embodiments, the desired biological functions of the
original toxin polypeptide from which the de-immunized CD8+ T-cell
hyper-immunized polypeptide was derived are preserved in the
polypeptides of the invention after the B-cell epitope(s) was
disrupted and the CD8+ T-cell epitope was added. In addition,
B-cell epitopes often coincide or overlap with epitopes of mature
CD4+ T-cells, thus the disruption of a B-cell epitope often
simultaneously disrupts a CD4+ T-cell epitope.
D. Selective Cytotoxicity Among Cell Types
[0319] Certain cell-targeted molecules of the present invention
have uses in the selective killing of specific target cells in the
presence of untargeted, bystander cells. By targeting the delivery
of immunogenic T-cell epitopes to the MHC class I pathway of target
cells, the subsequent presentation of T-cell epitopes and
CTL-mediated cytolysis of target cells induced by the cell-targeted
molecules of the invention can be restricted to preferentially
killing selected cell types in the presence of untargeted cells. In
addition, the killing of target cells by the potent cytotoxic
activity of various toxin effector regions can be restricted to
preferentially killing target cells with the simultaneous delivery
of an immunogenic T-cell epitope and a cytotoxic toxin effector
polypeptide.
[0320] In certain embodiments, upon administration of the
cell-targeted molecule of the present invention to a mixture of
cell types, the cell-targeted molecule is capable of selectively
killing those cells which are physically coupled with an
extracellular target biomolecule compared to cell types not
physically coupled with an extracellular target biomolecule.
Because many toxins are adapted for killing eukaryotic cells, such
as, e.g., members of the ABx and ribotoxin families, cytotoxic
proteins designed using toxin effector regions can show potent
cytotoxic activity. By targeting the delivery of enzymatically
active toxin effector regions to specific cell types using
high-affinity binding regions, this potent cell kill activity can
be restricted to killing only those cell types desired to be
targeted by their physical association with a target biomolecule of
the chosen binding regions.
[0321] In certain embodiments, the cytotoxic, cell-targeted
molecule of the present invention is capable of selectively or
preferentially causing the death of a specific cell type within a
mixture of two or more different cell types. This enables the
targeted cytotoxic activity to specific cell types with a high
preferentiality, such as a 3-fold cytotoxic effect, over
"bystander" cell types that do not express the target biomolecule.
Alternatively, the expression of the target biomolecule of the
binding region may be non-exclusive to one cell type if the target
biomolecule is expressed in low enough amounts and/or physically
coupled in low amounts with cell types that are not to be targeted.
This enables the targeted cell-killing of specific cell types with
a high preferentiality, such as a 3-fold cytotoxic effect, over
"bystander" cell types that do not express significant amounts of
the target biomolecule or are not physically coupled to significant
amounts of the target biomolecule.
[0322] In certain further embodiments, upon administration of the
cytotoxic cell-targeted molecule to two different populations of
cell types, the cytotoxic cell-targeted molecule is capable of
causing cell death as defined by the half-maximal cytotoxic
concentration (CD.sub.50) on a population of target cells, whose
members express an extracellular target biomolecule of the binding
region of the cytotoxic protein, at a dose at least three-times
lower than the CD.sub.50 dose of the same cytotoxic protein to a
population of cells whose members do not express an extracellular
target biomolecule of the binding region of the cytotoxic
protein.
[0323] In certain embodiments, the cytotoxic activity toward
populations of cell types physically coupled with an extracellular
target biomolecule is at least 3-fold higher than the cytotoxic
activity toward populations of cell types not physically coupled
with any extracellular target biomolecule of the binding region.
According to the present invention, selective cytotoxicity may be
quantified in terms of the ratio (a/b) of (a) cytotoxicity towards
a population of cells of a specific cell type physically coupled
with a target biomolecule of the binding region to (b) cytotoxicity
towards a population of cells of a cell type not physically coupled
with a target biomolecule of the binding region. In certain
embodiments, the cytotoxicity ratio is indicative of selective
cytotoxicity which is at least 3-fold, 5-fold, 10-fold, 15-fold,
20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold,
250-fold, 500-fold, 750-fold, or 1000-fold higher for populations
of cells or cell types physically coupled with a target biomolecule
of the binding region compared to populations of cells or cell
types not physically coupled with a target biomolecule of the
binding region.
[0324] This preferential cell-killing function allows a targeted
cell to be killed by certain cytotoxic, cell-targeted molecules of
the present invention under varied conditions and in the presence
of non-targeted bystander cells, such as ex vivo manipulated
mixtures of cell types, in vitro cultured tissues with mixtures of
cell types, or in vivo in the presence of multiple cell types (e.g.
in situ or in a native location within a multicellular
organism).
E. Delivery of Additional Exogenous Material into the Interior of
Targeted Cells
[0325] In addition to cytotoxic and cytostatic applications,
cell-targeted molecules of the present invention optionally may be
used for information gathering and diagnostic functions. Further,
non-toxic variants of the cytotoxic, cell-targeted molecules of the
invention, or optionally toxic variants, may be used to deliver
additional exogenous materials to and/or label the interiors of
cells physically coupled with an extracellular target biomolecule
of the cytotoxic protein. Various types of cells and/or cell
populations which express target biomolecules to at least one
cellular surface may be targeted by the cell-targeted molecules of
the invention for receiving exogenous materials. The functional
components of the present invention are modular, in that various
toxin effector regions and additional exogenous materials may be
linked to various binding regions to provide diverse applications,
such as non-invasive in vivo imaging of tumor cells.
[0326] Because the cell-targeted molecules of the invention,
including nontoxic forms thereof, are capable of entering cells
physically coupled with an extracellular target biomolecule
recognized by the cell-targeted molecule's binding region, certain
embodiments of the cell-targeted molecules of the invention may be
used to deliver additional exogenous materials into the interior of
targeted cell types. In one sense, the entire cell-targeted
molecule of the invention is an exogenous material which will enter
the cell; thus, the "additional" exogenous materials are
heterologous materials linked to but other than the core
cell-targeted molecule itself. CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptides which become non-toxic
after T-cell epitope addition may still be useful for delivering
exogenous materials into cells (e.g. T-cell epitope replacements
overlapping amino acid resides critical for catalytic function of a
toxin effector region).
[0327] "Additional exogenous material" as used herein refers to one
or more molecules, often not generally present within a native
target cell, where the proteins of the present invention can be
used to specifically transport such material to the interior of a
cell. Non-limiting examples of additional exogenous materials are
peptides, polypeptides, proteins, polynucleotides, small molecule
chemotherapeutic agents, and detection promoting agents.
[0328] In certain embodiments, the additional exogenous material
comprises a protein or polypeptide comprising an enzyme. In certain
other embodiments, the additional exogenous material is a nucleic
acid, such as, e.g. a ribonucleic acid that functions as a small
inhibiting RNA (siRNA) or microRNA (miRNA). In certain embodiments,
the additional exogenous material is an antigen, such as antigens
derived from bacterial proteins, viral proteins, proteins mutated
in cancer, proteins aberrantly expressed in cancer, or T-cell
complementary determining regions. For example, exogenous materials
include antigens, such as those characteristic of
antigen-presenting cells infected by bacteria, and T-cell
complementary determining regions capable of functioning as
exogenous antigens. Additional examples of exogenous materials
include polypeptides and proteins larger than an antigenic peptide,
such as enzymes. Exogenous materials comprising polypeptides or
proteins may optionally comprise one or more antigens whether known
or unknown to the skilled worker.
F. Information Gathering for Diagnostic Functions
[0329] Certain cell-targeted molecules of the present invention
have uses in the in vitro and/or in vivo detection of specific
cells, cell types, and/or cell populations. In certain embodiments,
the proteins described herein are used for both diagnosis and
treatment, or for diagnosis alone. When the same cytotoxic protein
is used for both diagnosis and treatment, the cytotoxic protein
variant which incorporates a detection promoting agent for
diagnosis may be rendered nontoxic by catalytic inactivation of a
toxin effector region via one or more amino acid substitutions,
including exemplary substitutions described herein. Nontoxic forms
of the cytotoxic, cell-targeted molecules of the invention that are
conjugated to detection promoting agents optionally may be used for
diagnostic functions, such as for companion diagnostics used in
conjunction with a therapeutic regimen comprising the same or a
related binding region.
[0330] The ability to conjugate detection promoting agents known in
the art to various cell-targeted molecules of the present invention
provides useful compositions for the detection of cancer, tumor,
immune, and infected cells. These diagnostic embodiments of the
cell-targeted molecules of the invention may be used for
information gathering via various imaging techniques and assays
known in the art. For example, diagnostic embodiments of the
cell-targeted molecules of the invention may be used for
information gathering via imaging of intracellular organelles (e.g.
endocytotic, Golgi, endoplasmic reticulum, and cytosolic
compartments) of individual cancer cells, immune cells, or infected
cells in a patient or biopsy sample.
[0331] Various types of information may be gathered using the
diagnostic embodiments of the cell-targeted molecules of the
invention whether for diagnostic uses or other uses. This
information may be useful, for example, in diagnosing neoplastic
cell types, determining therapeutic susceptibilities of a patient's
disease, assaying the progression of anti-neoplastic therapies over
time, assaying the progression of immunomodulatory therapies over
time, assaying the progression of antimicrobial therapies over
time, evaluating the presence of infected cells in transplantation
materials, evaluating the presence of unwanted cell types in
transplantation materials, and/or evaluating the presence of
residual tumor cells after surgical excision of a tumor mass.
[0332] For example, subpopulations of patients might be ascertained
using information gathered using the diagnostic variants of the
cell-targeted molecules of the invention, and then individual
patients could be categorized into subpopulations based on their
unique characteristic(s) revealed using those diagnostic
embodiments. For example, the effectiveness of specific
pharmaceuticals or therapies might be one type of criterion used to
define a patient subpopulation. For example, a nontoxic diagnostic
variant of a particular cytotoxic, cell-targeted molecule of the
invention may be used to differentiate which patients are in a
class or subpopulation of patients predicted to respond positively
to a cytotoxic variant of the same cell-targeted molecule of the
invention. Accordingly, associated methods for patient
identification, patient stratification, and diagnosis using CD8+
T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
cell-targeted molecules of the present invention, including
non-toxic variants of cytotoxic, cell-targeted molecules of the
present invention, are considered to be within the scope of the
present invention.
VII. Production, Manufacture, and Purification of CD8+ T-Cell
Hyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized Polypeptides
of the Present Invention and the Cell-Targeted Molecules Comprising
the Same
[0333] The CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptides and cell-targeted molecules of the
present invention may be produced using biochemical engineering
techniques well known to those of skill in the art. For example,
polypeptides and cell-targeted molecules of the invention may be
manufactured by standard synthetic methods, by use of recombinant
expression systems, or by any other suitable method. Thus,
polypeptides and cell-targeted proteins of the present invention
may be synthesized in a number of ways, including, e.g. methods
comprising: (1) synthesizing a polypeptide or polypeptide component
of a protein using standard solid-phase or liquid-phase
methodology, either stepwise or by fragment assembly, and isolating
and purifying the final peptide compound product; (2) expressing a
polynucleotide that encodes a polypeptide or polypeptide component
of a cell-targeted protein of the invention in a host cell and
recovering the expression product from the host cell or host cell
culture; or (3) cell-free in vitro expression of a polynucleotide
encoding a polypeptide or polypeptide component of a cell-targeted
protein of the invention, and recovering the expression product; or
by any combination of the methods of (1), (2) or (3) to obtain
fragments of the peptide component, subsequently joining (e.g.
ligating) the fragments to obtain the peptide component, and
recovering the peptide component.
[0334] It may be preferable to synthesize a CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide
or a protein or polypeptide component of a cell-targeted protein of
the invention by means of solid-phase or liquid-phase peptide
synthesis. Polypeptides and cell-targeted molecules of the present
invention may suitably be manufactured by standard synthetic
methods. Thus, peptides may be synthesized by, e.g. methods
comprising synthesizing the peptide by standard solid-phase or
liquid-phase methodology, either stepwise or by fragment assembly,
and isolating and purifying the final peptide product. In this
context, reference may be made to WO 1998/11125 or, inter alia,
Fields G et al., Principles and Practice of Solid-Phase Peptide
Synthesis (Synthetic Peptides, Grant G, ed., Oxford University
Press, U.K., 2nd ed., 2002) and the synthesis examples therein.
[0335] CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptides and cytotoxic, cell-targeted proteins of
the present invention may be prepared (produced and purified) using
recombinant techniques well known in the art. In general, methods
for preparing polypeptides by culturing host cells transformed or
transfected with a vector comprising the encoding polynucleotide
and recovering the polypeptide from cell culture are described in,
e.g. Sambrook J et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, NY, U.S., 1989); Dieffenbach
C et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, N.Y., U.S., 1995). Any suitable host cell may be
used to produce a polypeptide and/or cell-targeted protein of the
invention. Host cells may be cells stably or transiently
transfected, transformed, transduced or infected with one or more
expression vectors which drive expression of a polypeptide of the
invention. In addition, a CD8+ T-cell hyper-immunized and/or
B-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targeted
protein of the invention may be produced by modifying the
polynucleotide encoding a polypeptide or cell-targeted protein of
the invention that result in altering one or more amino acids or
deleting or inserting one or more amino acids in order to achieve
desired properties, such as changed cytotoxicity, changed
cytostatic effects, and/or changed serum half-life.
[0336] There are a wide variety of expression systems which may be
chosen to produce a polypeptide or cell-targeted protein of the
present invention. For example, host organisms for expression of
cell-targeted proteins of the invention include prokaryotes, such
as E. coli and B. subtilis, eukaryotic cells, such as yeast and
filamentous fungi (like S. cerevisiae, P. pastoris, A. awamori, and
K. lactis), algae (like C. reinhardtii), insect cell lines,
mammalian cells (like CHO cells), plant cell lines, and eukaryotic
organisms such as transgenic plants (like A. thaliana and N.
benthamiana).
[0337] Accordingly, the present invention also provides methods for
producing a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptide and/or cell-targeted protein of the
invention according to above recited methods and using a
polynucleotide encoding part or all of a polypeptide of the
invention or a polypeptide component of a cell-targeted protein of
the invention, an expression vector comprising at least one
polynucleotide of the invention capable of encoding part or all of
a polypeptide of the invention when introduced into a host cell,
and/or a host cell comprising a polynucleotide or expression vector
of the invention.
[0338] When a polypeptide or protein is expressed using recombinant
techniques in a host cell or cell-free system, it is advantageous
to separate (or purify) the desired polypeptide or protein away
from other components, such as host cell factors, in order to
obtain preparations that are of high purity or are substantially
homogeneous. Purification can be accomplished by methods well known
in the art, such as centrifugation techniques, extraction
techniques, chromatographic and fractionation techniques (e.g. size
separation by gel filtration, charge separation by ion-exchange
column, hydrophobic interaction chromatography, reverse phase
chromatography, chromatography on silica or cation-exchange resins
such as DEAE and the like, chromatofocusing, and Protein A
Sepharose chromatography to remove contaminants), and precipitation
techniques (e.g. ethanol precipitation or ammonium sulfate
precipitation). Any number of biochemical purification techniques
may be used to increase the purity of a CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptide and/or
cell-targeted molecule of the present invention. In certain
embodiments, the polypeptides and cell-targeted molecules of the
invention may optionally be purified in homo-multimeric forms (i.e.
a protein complex of two or more identical polypeptides or
cell-targeted molecules of the invention).
[0339] In the Examples below are descriptions of non-limiting
examples of methods for producing a polypeptide or cell-targeted
molecule of the invention, as well as specific but non-limiting
aspects of production for exemplary cell-targeted molecules of the
present invention.
VIII. Pharmaceutical and Diagnostic Compositions Comprising a
T-Cell Hyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized
Polypeptide of the Present Invention or Cell-Targeted Molecule
Comprising the Same
[0340] The present invention provides polypeptides and proteins for
use, alone or in combination with one or more additional
therapeutic agents, in a pharmaceutical composition, for treatment
or prophylaxis of conditions, diseases, disorders, or symptoms
described in further detail below (e.g. cancers, malignant tumors,
non-malignant tumors, growth abnormalities, immune disorders, and
microbial infections). The present invention further provides
pharmaceutical compositions comprising a polypeptide or
cell-targeted molecule of the invention, or a pharmaceutically
acceptable salt or solvate thereof, according to the invention,
together with at least one pharmaceutically acceptable carrier,
excipient, or vehicle. In certain embodiments, the pharmaceutical
composition of the present invention may comprise homo-multimeric
and/or hetero-multimeric forms of the polypeptides or cell-targeted
molecules of the invention. The pharmaceutical compositions will be
useful in methods of treating, ameliorating, or preventing a
disease, condition, disorder, or symptom described in further
detail below. Each such disease, condition, disorder, or symptom is
envisioned to be a separate embodiment with respect to uses of a
pharmaceutical composition according to the invention. The
invention further provides pharmaceutical compositions for use in
at least one method of treatment according to the invention, as
described in more detail below.
[0341] As used herein, the terms "patient" and "subject" are used
interchangeably to refer to any organism, commonly vertebrates such
as humans and animals, which presents symptoms, signs, and/or
indications of at least one disease, disorder, or condition. These
terms include mammals such as the non-limiting examples of
primates, livestock animals (e.g. cattle, horses, pigs, sheep,
goats, etc.), companion animals (e.g. cats, dogs, etc.) and
laboratory animals (e.g. mice, rabbits, rats, etc.).
[0342] As used herein, "treat," "treating," or "treatment" and
grammatical variants thereof refer to an approach for obtaining
beneficial or desired clinical results. The terms may refer to
slowing the onset or rate of development of a condition, disorder
or disease, reducing or alleviating symptoms associated with it,
generating a complete or partial regression of the condition, or
some combination of any of the above. For the purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, reduction or alleviation of symptoms, diminishment
of extent of disease, stabilization (e.g. not worsening) of state
of disease, delay or slowing of disease progression, amelioration
or palliation of the disease state, and remission (whether partial
or total), whether detectable or undetectable. "Treat," "treating,"
or "treatment" can also mean prolonging survival relative to
expected survival time if not receiving treatment. A subject (e.g.
a human) in need of treatment may thus be a subject already
afflicted with the disease or disorder in question. The terms
"treat," "treating," or "treatment" includes inhibition or
reduction of an increase in severity of a pathological state or
symptoms relative to the absence of treatment, and is not
necessarily meant to imply complete cessation of the relevant
disease, disorder, or condition. With regard to tumors and/or
cancers, treatment includes reduction in overall tumor burden
and/or individual tumor size.
[0343] As used herein, the terms "prevent," "preventing,"
"prevention" and grammatical variants thereof refer to an approach
for preventing the development of, or altering the pathology of, a
condition, disease, or disorder. Accordingly, "prevention" may
refer to prophylactic or preventive measures. For the purposes of
this invention, beneficial or desired clinical results include, but
are not limited to, prevention or slowing of symptoms, progression
or development of a disease, whether detectable or undetectable. A
subject (e.g. a human) in need of prevention may thus be a subject
not yet afflicted with the disease or disorder in question. The
term "prevention" includes slowing the onset of disease relative to
the absence of treatment, and is not necessarily meant to imply
permanent prevention of the relevant disease, disorder or
condition. Thus "preventing" or "prevention" of a condition may in
certain contexts refer to reducing the risk of developing the
condition, or preventing or delaying the development of symptoms
associated with the condition.
[0344] As used herein, an "effective amount" or "therapeutically
effective amount" is an amount or dose of a composition (e.g. a
therapeutic composition or agent) that produces at least one
desired therapeutic effect in a subject, such as preventing or
treating a target condition or beneficially alleviating a symptom
associated with the condition. The most desirable therapeutically
effective amount is an amount that will produce a desired efficacy
of a particular treatment selected by one of skill in the art for a
given subject in need thereof. This amount will vary depending upon
a variety of factors understood by the skilled worker, including
but not limited to the characteristics of the therapeutic compound
(including activity, pharmacokinetics, pharmacodynamics, and
bioavailability), the physiological condition of the subject
(including age, sex, disease type, disease stage, general physical
condition, responsiveness to a given dosage, and type of
medication), the nature of the pharmaceutically acceptable carrier
or carriers in the formulation, and the route of administration.
One skilled in the clinical and pharmacological arts will be able
to determine a therapeutically effective amount through routine
experimentation, namely by monitoring a subject's response to
administration of a compound and adjusting the dosage accordingly
(see e.g. Remington: The Science and Practice of Pharmacy (Gennaro
A, ed., Mack Publishing Co., Easton, Pa., U.S., 19th ed.,
1995)).
[0345] Diagnostic compositions comprise a polypeptide or
cell-targeted molecule of the invention and one or more detection
promoting agents. Various detection promoting agents are known in
the art, such as isotopes, dyes, colorimetric agents, contrast
enhancing agents, fluorescent agents, bioluminescent agents, and
magnetic agents. These agents may be incorporated into the
polypeptide or cell-targeted molecule of the invention at any
position. The incorporation of the agent may be via an amino acid
residue(s) of the protein or via some type of linkage known in the
art, including via linkers and/or chelators. The incorporation of
the agent is in such a way to enable the detection of the presence
of the diagnostic composition in a screen, assay, diagnostic
procedure, and/or imaging technique.
[0346] When producing or manufacturing a diagnostic composition of
the present invention, a cell-targeted molecule of the invention
may be directly or indirectly linked to one or more detection
promoting agents. There are numerous detection promoting agents
known to the skilled worker which can be operably linked to the
polypeptides or cell-targeted molecules of the invention for
information gathering methods, such as for diagnostic and/or
prognostic applications to diseases, disorders, or conditions of an
organism (see e.g. Cai W et al., J Nucl Med 48: 304-10 (2007);
Nayak T, Brechbiel M, Bioconjug Chem 20: 825-41 (2009); Paudyal P
et al., Oncol Rep 22: 115-9 (2009); Qiao J et al., PLoS ONE 6:
e18103 (2011); Sano K et al., Breast Cancer Res 14: R61 (2012)).
For example, detection promoting agents include image enhancing
contrast agents, such as fluorescent dyes (e.g. Alexa680,
indocyanine green, and Cy5.5), isotopes and radionuclides, such as
.sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.32P, .sup.51Mn,
.sup.52mMn, .sup.52Fe, .sup.55Co, .sup.62Cu, .sup.64Cu, .sup.67Cu,
.sup.67Ga, .sup.68Ga, .sup.72As, .sup.73Se, .sup.75Br, .sup.76Br,
.sup.82mRb, .sup.83Sr, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.94mTc,
.sup.94Tc, .sup.99mTc, .sup.110In, .sup.111In, .sup.120I,
.sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.154Gd, .sup.155Gd,
.sup.156Gd, .sup.157Gd, .sup.158Gd, .sup.177Lu, .sup.186Re,
.sup.188Re, and .sup.223R; paramagnetic ions, such as chromium
(III), manganese (II), iron (III), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) or erbium (III); metals, such as lanthanum
(III), gold (III), lead (II), and bismuth (III);
ultrasound-contrast enhancing agents, such as liposomes; radiopaque
agents, such as barium, gallium, and thallium compounds. Detection
promoting agents may be incorporated directly or indirectly by
using an intermediary functional group, such as chelators like
2-benzyl DTPA, PAMAM, NOTA, DOTA, TETA, analogs thereof, and
functional equivalents of any of the foregoing (see Leyton J et
al., Clin Cancer Res 14: 7488-96 (2008)).
[0347] There are numerous standard techniques known to the skilled
worker for incorporating, affixing, and/or conjugating various
detection promoting agents to proteins, especially to
immunoglobulins and immunoglobulin-derived domains (Wu A, Methods
65: 139-47 (2014)). Similarly, there are numerous imaging
approaches known to the skilled worker, such as non-invasive in
vivo imaging techniques commonly used in the medical arena, for
example: computed tomography imaging (CT scanning), optical imaging
(including direct, fluorescent, and bioluminescent imaging),
magnetic resonance imaging (MRI), positron emission tomography
(PET), single-photon emission computed tomography (SPECT),
ultrasound, and x-ray computed tomography imaging (see Kaur S et
al., Cancer Lett 315: 97-111 (2012), for review).
IX. Production or Manufacture of a Pharmaceutical and/or Diagnostic
Composition Comprising a T-Cell Hyper-Immunized and/or B-Cell/CD4+
T-Cell De-Immunized Polypeptide or Cell-Targeted Molecule of the
Present Invention
[0348] Pharmaceutically acceptable salts or solvates of any of the
polypeptides and cell-targeted molecules of the invention are
likewise within the scope of the present invention.
[0349] The term "solvate" in the context of the present invention
refers to a complex of defined stoichiometry formed between a
solute (in casu, a polypeptide compound or pharmaceutically
acceptable salt thereof according to the invention) and a solvent.
The solvent in this connection may, for example, be water, ethanol
or another pharmaceutically acceptable, typically small-molecular
organic species, such as, but not limited to, acetic acid or lactic
acid. When the solvent in question is water, such a solvate is
normally referred to as a hydrate.
[0350] Polypeptides and proteins of the present invention, or salts
thereof, may be formulated as pharmaceutical compositions prepared
for storage or administration, which typically comprise a
therapeutically effective amount of a compound of the present
invention, or a salt thereof, in a pharmaceutically acceptable
carrier. The term "pharmaceutically acceptable carrier" includes
any of the standard pharmaceutical carriers. Pharmaceutically
acceptable carriers for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro, ed.,
1985). As used herein, "pharmaceutically acceptable carrier"
includes any and all physiologically acceptable, i.e. compatible,
solvents, dispersion media, coatings, antimicrobial agents,
isotonic, and absorption delaying agents, and the like.
Pharmaceutically acceptable carriers or diluents include those used
in formulations suitable for oral, rectal, nasal or parenteral
(including subcutaneous, intramuscular, intravenous, intradermal,
and transdermal) administration. Exemplary pharmaceutically
acceptable carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. Examples of
suitable aqueous and nonaqueous carriers that may be employed in
the pharmaceutical compositions of the invention include water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as
ethyloleate. Proper fluidity can be maintained, for example, by the
use of coating materials, such as lecithin, by the maintenance of
the required particle size in the case of dispersions, and by the
use of surfactants. In certain embodiments, the carrier is suitable
for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g. by injection or infusion). Depending
on selected route of administration, the protein or other
pharmaceutical component may be coated in a material intended to
protect the compound from the action of low pH and other natural
inactivating conditions to which the active protein may encounter
when administered to a patient by a particular route of
administration.
[0351] The formulations of the pharmaceutical compositions of the
invention may conveniently be presented in unit dosage form and may
be prepared by any of the methods well known in the art of
pharmacy. In such form, the composition is divided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of the preparations, for example, packeted
tablets, capsules, and powders in vials or ampoules. The unit
dosage form can also be a capsule, cachet, or tablet itself, or it
can be the appropriate number of any of these packaged forms. It
may be provided in single dose injectable form, for example in the
form of a pen. Compositions may be formulated for any suitable
route and means of administration. Subcutaneous or transdermal
modes of administration may be particularly suitable for
therapeutic proteins described herein.
[0352] The pharmaceutical compositions of the present invention may
also contain adjuvants such as preservatives, wetting agents,
emulsifying agents and dispersing agents. Preventing the presence
of microorganisms may be ensured both by sterilization procedures,
and by the inclusion of various antibacterial and antifungal
agents, for example, paraben, chlorobutanol, phenol sorbic acid,
and the like. Isotonic agents, such as sugars, sodium chloride, and
the like into the compositions, may also be desirable. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as, aluminum monostearate and gelatin.
[0353] A pharmaceutical composition of the present invention also
optionally includes a pharmaceutically acceptable antioxidant.
Exemplary pharmaceutically acceptable antioxidants are water
soluble antioxidants such as ascorbic acid, cysteine hydrochloride,
sodium bisulfate, sodium metabisulfite, sodium sulfite and the
like; oil-soluble antioxidants, such as ascorbyl palmitate,
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
lecithin, propylgallate, alpha-tocopherol, and the like; and metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0354] In another aspect, the present invention provides
pharmaceutical compositions comprising one or a combination of
different polypeptides and/or cell-targeted molecules of the
invention, or an ester, salt or amide of any of the foregoing, and
at least one pharmaceutically acceptable carrier.
[0355] Therapeutic compositions are typically sterile and stable
under the conditions of manufacture and storage. The composition
may be formulated as a solution, microemulsion, liposome, or other
ordered structure suitable to high drug concentration. The carrier
may be a solvent or dispersion medium containing, for example,
water, alcohol such as ethanol, polyol (e.g. glycerol, propylene
glycol, and liquid polyethylene glycol), or any suitable mixtures.
The proper fluidity may be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by use of surfactants
according to formulation chemistry well known in the art. In
certain embodiments, isotonic agents, e.g. sugars, polyalcohols
such as mannitol, sorbitol, or sodium chloride may be desirable in
the composition. Prolonged absorption of injectable compositions
may be brought about by including in the composition an agent that
delays absorption for example, monostearate salts and gelatin.
[0356] Solutions or suspensions used for intradermal or
subcutaneous application typically include one or more of: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates; and
tonicity adjusting agents such as, e.g., sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide, or buffers with citrate,
phosphate, acetate and the like. Such preparations may be enclosed
in ampoules, disposable syringes or multiple dose vials made of
glass or plastic.
[0357] Sterile injectable solutions may be prepared by
incorporating a polypeptide or cell-targeted molecule of the
invention in the required amount in an appropriate solvent with one
or a combination of ingredients described above, as required,
followed by sterilization microfiltration. Dispersions may be
prepared by incorporating the active compound into a sterile
vehicle that contains a dispersion medium and other ingredients,
such as those described above. In the case of sterile powders for
the preparation of sterile injectable solutions, the methods of
preparation are vacuum drying and freeze-drying (lyophilization)
that yield a powder of the active ingredient in addition to any
additional desired ingredient from a sterile-filtered solution
thereof.
[0358] When a therapeutically effective amount of a polypeptide or
cell-targeted molecule of the invention is designed to be
administered by, e.g. intravenous, cutaneous or subcutaneous
injection, the binding agent will be in the form of a pyrogen-free,
parenterally acceptable aqueous solution. Methods for preparing
parenterally acceptable protein solutions, taking into
consideration appropriate pH, isotonicity, stability, and the like,
are within the skill in the art. A preferred pharmaceutical
composition for intravenous, cutaneous, or subcutaneous injection
will contain, in addition to binding agents, an isotonic vehicle
such as sodium chloride injection, Ringer's injection, dextrose
injection, dextrose and sodium chloride injection, lactated
Ringer's injection, or other vehicle as known in the art. A
pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additives well known to those of skill in the art.
[0359] As described elsewhere herein, a polypeptide or
cell-targeted molecule of the invention may be prepared with
carriers that will protect the compound against rapid release, such
as a controlled release formulation, including implants,
transdermal patches, and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Many methods for the
preparation of such formulations are patented or generally known to
those skilled in the art (see e.g. Sustained and Controlled Release
Drug Delivery Systems (Robinson J, ed., Marcel Dekker, Inc., NY,
U.S., 1978)).
[0360] In certain embodiments, the pharmaceutical composition of
the present invention may be formulated to ensure a desired
distribution in vivo. For example, the blood-brain barrier excludes
many large and/or hydrophilic compounds. To target a therapeutic
compound or composition of the invention to a particular in vivo
location, they can be formulated, for example, in liposomes which
may comprise one or more moieties that are selectively transported
into specific cells or organs, thus enhancing targeted drug
delivery. Exemplary targeting moieties include folate or biotin;
mannosides; antibodies; surfactant protein A receptor; p120 catenin
and the like.
[0361] Pharmaceutical compositions include parenteral formulations
designed to be used as implants or particulate systems. Examples of
implants are depot formulations composed of polymeric or
hydrophobic components such as emulsions, ion exchange resins, and
soluble salt solutions. Examples of particulate systems are
microspheres, microparticles, nanocapsules, nanospheres, and
nanoparticles (see e.g. Honda M et al., Int J Nanomedicine 8:
495-503 (2013); Sharma A et al., Biomed Res Int 2013: 960821
(2013); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012)).
Controlled release formulations may be prepared using polymers
sensitive to ions, such as, e.g. liposomes, polaxamer 407, and
hydroxyapatite.
X. Polynucleotides, Expression Vectors, and Host Cells
[0362] Beyond the polypeptides and proteins of the present
invention, the polynucleotides that encode the polypeptides and
cell-targeted molecules of the invention, or functional portions
thereof, are also encompassed within the scope of the present
invention. The term "polynucleotide" is equivalent to the term
"nucleic acid," each of which includes one or more of: polymers of
deoxyribonucleic acids (DNAs), polymers of ribonucleic acids
(RNAs), analogs of these DNAs or RNAs generated using nucleotide
analogs, and derivatives, fragments and homologs thereof. The
polynucleotide of the present invention may be single-, double-, or
triple-stranded. Such polynucleotides are specifically disclosed to
include all polynucleotides capable of encoding an exemplary
protein, for example, taking into account the wobble known to be
tolerated in the third position of RNA codons, yet encoding for the
same amino acid as a different RNA codon (see Stothard P,
Biotechniques 28: 1102-4 (2000)).
[0363] In one aspect, the invention provides polynucleotides which
encode a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptide and/or cell-targeted protein of the
invention, or a fragment or derivative thereof. The polynucleotides
may include, e.g., nucleic acid sequence encoding a polypeptide at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or
more, identical to a polypeptide comprising one of the amino acid
sequences of the protein. The invention also includes
polynucleotides comprising nucleotide sequences that hybridize
under stringent conditions to a polynucleotide which encodes CD8+
T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
polypeptide and/or cell-targeted protein of the invention, or a
fragment or derivative thereof, or the antisense or complement of
any such sequence.
[0364] Derivatives or analogs of the molecules (e.g., CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides
and/or cell-targeted proteins comprising the same) of the present
invention include, inter alia, polynucleotide (or polypeptide)
molecules having regions that are substantially homologous to the
polynucleotides, CD8+ T-cell hyper-immunized and/or B-cell/CD4+
T-cell de-immunized polypeptides, or cell-targeted proteins of the
present invention, e.g. by at least about 45%, 50%, 70%, 80%, 95%,
98%, or even 99% identity (with a preferred identity of 80-99%)
over a polynucleotide or polypeptide sequence of the same size or
when compared to an aligned sequence in which the alignment is done
by a computer homology program known in the art. An exemplary
program is the GAP program (Wisconsin Sequence Analysis Package,
Version 8 for UNIX, Genetics Computer Group, University Research
Park, Madison, Wis., U.S.) using the default settings, which uses
the algorithm of Smith T, Waterman M, Adv. Appl. Math. 2: 482-9
(1981). Also included are polynucleotides capable of hybridizing to
the complement of a sequence encoding the cell-targeted proteins of
the invention under stringent conditions (see e.g. Ausubel F et
al., Current Protocols in Molecular Biology (John Wiley & Sons,
New York, N.Y., U.S., 1993)), and below. Stringent conditions are
known to those skilled in the art and may be found, e.g., in
Current Protocols in Molecular Biology (John Wiley & Sons, NY,
U.S., Ch. Sec. 6.3.1-6.3.6 (1989)).
[0365] The present invention further provides expression vectors
that comprise the polynucleotides within the scope of the present
invention. The polynucleotides capable of encoding the CD8+ T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides
and/or cell-targeted proteins of the invention may be inserted into
known vectors, including bacterial plasmids, viral vectors and
phage vectors, using material and methods well known in the art to
produce expression vectors. Such expression vectors will include
the polynucleotides necessary to support production of contemplated
CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized
polypeptides and/or cell-targeted proteins of the invention within
any host cell of choice or cell-free expression systems (e.g. pTxb1
and pIVEX2.3). The specific polynucleotides comprising expression
vectors for use with specific types of host cells or cell-free
expression systems are well known to one of ordinary skill in the
art, can be determined using routine experimentation, or may be
purchased.
[0366] The term "expression vector," as used herein, refers to a
polynucleotide, linear or circular, comprising one or more
expression units. The term "expression unit" denotes a
polynucleotide segment encoding a polypeptide of interest and
capable of providing expression of the nucleic acid segment in a
host cell. An expression unit typically comprises a transcription
promoter, an open reading frame encoding the polypeptide of
interest, and a transcription terminator, all in operable
configuration. An expression vector contains one or more expression
units. Thus, in the context of the present invention, an expression
vector encoding a CD8+ T-cell hyper-immunized and/or B-cell/CD4+
T-cell de-immunized polypeptide and/or protein comprising a single
polypeptide chain (e.g. a scFv genetically recombined with a CD8+
T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga
toxin effector region) includes at least an expression unit for the
single polypeptide chain, whereas a protein comprising, e.g. two or
more polypeptide chains (e.g. one chain comprising a V.sub.L domain
and a second chain comprising a V.sub.H domain linked to a toxin
effector region) includes at least two expression units, one for
each of the two polypeptide chains of the protein. For expression
of multi-chain cell-targeted proteins of the invention, an
expression unit for each polypeptide chain may also be separately
contained on different expression vectors (e.g. expression may be
achieved with a single host cell into which expression vectors for
each polypeptide chain has been introduced).
[0367] Expression vectors capable of directing transient or stable
expression of polypeptides and proteins are well known in the art.
The expression vectors generally include, but are not limited to,
one or more of the following: a heterologous signal sequence or
peptide, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence, each of which is well known in the art. Optional
regulatory control sequences, integration sequences, and useful
markers that can be employed are known in the art.
[0368] The term "host cell" refers to a cell which can support the
replication or expression of the expression vector. Host cells may
be prokaryotic cells, such as E. coli or eukaryotic cells (e.g.
yeast, insect, amphibian, bird, or mammalian cells). Creation and
isolation of host cell lines comprising a polynucleotide of the
invention or capable of producing a polypeptide and/or
cell-targeted protein of the invention can be accomplished using
standard techniques known in the art.
[0369] CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptides and/or proteins within the scope of the
present invention may be variants or derivatives of the
polypeptides and proteins described herein that are produced by
modifying the polynucleotide encoding a polypeptide and/or protein
by altering one or more amino acids or deleting or inserting one or
more amino acids that may render it more suitable to achieve
desired properties, such as more optimal expression by a host
cell.
XI. Delivery Devices and Kits
[0370] In certain embodiments, the invention relates to a device
comprising one or more compositions of matter of the invention,
such as a pharmaceutical composition, for delivery to a subject in
need thereof. Thus, a delivery device comprising one or more
compounds of the invention can be used to administer to a patient a
composition of matter of the invention by various delivery methods,
including: intravenous, subcutaneous, intramuscular or
intraperitoneal injection; oral administration; transdermal
administration; pulmonary or transmucosal administration;
administration by implant, osmotic pump, cartridge or micro pump;
or by other means recognized by a person of skill in the art.
[0371] Also within the scope of the invention are kits comprising
at least one composition of matter of the invention, and
optionally, packaging and instructions for use. Kits may be useful
for drug administration and/or diagnostic information gathering. A
kit of the invention may optionally comprise at least one
additional reagent (e.g., standards, markers and the like). Kits
typically include a label indicating the intended use of the
contents of the kit. The kit may further comprise reagents and
other tools for detecting a cell type (e.g. tumor cell) in a sample
or in a subject, or for diagnosing whether a patient belongs to a
group that responds to a therapeutic strategy which makes use of a
compound, composition or related method of the invention as
described herein.
XII. Methods of Generating T-Cell Hyper-Immunized and/or
B-Cell/CD4+ T-Cell De-Immunized Polypeptides of the Present
Invention
[0372] The present invention provides methods of creating T-cell
hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides
of the present invention by modifying polypeptides already capable
of intracellularly routing to a cytosol, ER, or lysosome of a cell
from an endosomal compartment of the cell; the method comprising
the step of adding a heterologous T-cell epitope to the
polypeptide. In certain further methods of the present invention,
the heterologous T-cell epitope is embedded or inserted within a
polypeptide capable of intracellularly routing to a cytosol, ER, or
lysosome of a cell from an endosomal compartment of the cell.
[0373] In certain embodiments of the methods of the present
invention, a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptide of the present invention is created by
modifying a polypeptide already capable of intracellularly routing
to a cytosol, ER, or lysosome of a cell from an endosomal
compartment of the cell; the method comprising the step of adding a
heterologous T-cell epitope to the polypeptide. In certain further
methods of the present invention, the heterologous T-cell epitope
is embedded or inserted within a polypeptide capable of
intracellularly routing to a cytosol, ER, or lysosome of a cell
from an endosomal compartment of the cell.
[0374] In certain embodiments of the methods of the present
invention, a polypeptide already capable of intracellularly routing
to a cytosol, ER, or lysosome of a cell from an endosomal
compartment of the cell is created into a T-cell hyper-immunized
polypeptide of the present invention; the method comprising the
step of adding a heterologous T-cell epitope to the polypeptide. In
certain further embodiments of the methods of the present
invention, a polypeptide already capable of intracellularly routing
to a cytosol, ER, or lysosome of a cell from an endosomal
compartment of the cell is created into a CD8+ T-cell
hyper-immunized polypeptide of the present invention; the method
comprising the step of adding a heterologous T-cell epitope to the
polypeptide. In certain further methods of the present invention,
the heterologous T-cell epitope is embedded or inserted within a
polypeptide capable of intracellularly routing to a cytosol, ER, or
lysosome of a cell from an endosomal compartment of the cell.
[0375] In certain embodiments of the methods of the present
invention, a polypeptide capable of delivering a T-cell epitope for
presentation by a MHC class I molecule is created; the method
comprising the step of adding a heterologous T-cell epitope to a
polypeptide capable of intracellular delivery of the T-cell epitope
from an endosomal compartment of a cell to a proteasome of the
cell. In certain further methods of the present invention, the
heterologous T-cell epitope is embedded or inserted within a
polypeptide capable of intracellularly routing to a cytosol, ER, or
lysosome of a cell from an endosomal compartment of the cell.
[0376] In certain embodiments of the methods of the present
invention, a T-cell hyper-immunized and/or B-cell/CD4+ T-cell
de-immunized polypeptide is created; the method comprising the step
of inserting or embedding a heterologous T-cell epitope into an
endogenous B-cell epitope region of a polypeptide already capable
of intracellularly routing to a cytosol, ER, or lysosome of a cell
from an endosomal compartment of the cell.
[0377] In certain embodiments of the methods of the present
invention, a CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cell
de-immunized polypeptide of the present invention is created; the
method comprising the step of embedding or inserting a heterologous
T-cell epitope into an endogenous B-cell epitope region of a
polypeptide already capable of intracellularly routing to a
cytosol, ER, or lysosome of a cell from an endosomal compartment of
the cell.
[0378] In certain embodiments of the methods of the present
invention, a polypeptide already capable of intracellularly routing
to a cytosol, ER, or lysosome of a cell from an endosomal
compartment of the cell is created into a T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptide of the present
invention; the method comprising the step of embedding or inserting
a heterologous T-cell epitope into an endogenous B-cell epitope
region of the polypeptide. In certain further embodiments of the
methods of the present invention, a polypeptide already capable of
intracellularly routing to a cytosol, ER, or lysosome of a cell
from an endosomal compartment of the cell is created into a CD8+
T-cell hyper-immunized polypeptide of the present invention; the
method comprising the step of embedding or inserting a heterologous
T-cell epitope into an endogenous B-cell epitope region of the
polypeptide.
[0379] In certain embodiments of the methods of the present
invention, a de-immunized polypeptide capable of delivering a
T-cell epitope for presentation by a MHC class I molecule is
created; the method comprising the step of embedding or inserting a
heterologous T-cell epitope into an endogenous B-cell epitope
region of a polypeptide capable of intracellular delivery of the
T-cell epitope from an endosomal compartment of a cell to a
proteasome of the cell.
[0380] In certain embodiments of the methods of the present
invention, a de-immunized polypeptide is created which has reduced
B-cell immunogenicity when administered to a chordate. In certain
embodiments of the methods of the present invention, is a method
for reducing B-cell immunogenicity in a polypeptide, the method
comprising the step of disrupting a B-cell epitope region within a
polypeptide with one or more amino acid residue(s) comprised by a
heterologous T-cell epitope added to the polypeptide. In certain
further embodiments, the disrupting step further comprises creating
one or more amino acid substitutions in the B-cell epitope region.
In certain further embodiments, the disrupting step further
comprises creating one or more amino acid insertions in the B-cell
epitope region.
[0381] Certain embodiments of the methods of the present invention
are methods for reducing B-cell immunogenicity in a polypeptide
while simultaneously increasing CD8+ T-cell immunogenicity after
administration to a chordate, the methods comprising the step of
disrupting a B-cell epitope region within a polypeptide with one or
more amino acid residue(s) comprised by a heterologous CD8+ T-cell
epitope added to the polypeptide. In certain further embodiments,
the disrupting step further comprises creating one or more amino
acid substitutions in the B-cell epitope region. In certain further
embodiments, the disrupting step further comprises creating one or
more amino acid insertions in the B-cell epitope region.
[0382] Certain embodiments of the methods of the present invention
are methods for reducing B-cell immunogenicity in a polypeptide
while simultaneously increasing CD8+ T-cell immunogenicity after
administration to a chordate, the methods comprising the steps of:
1) identifying a B-cell epitope in a polypeptide; and 2) disrupting
the identified B-cell epitope with one or more amino acid
residue(s) comprised by a heterologous CD8+ T-cell epitope added to
the polypeptide. In certain further embodiments, the disrupting
step further comprises the creation of one or more amino acid
substitutions in the B-cell epitope region. In certain further
embodiments, the disrupting step further comprises creating one or
more amino acid insertions in the B-cell epitope region.
[0383] Certain embodiments of the methods of the present invention
are methods for reducing B-cell immunogenicity in a polypeptide
while simultaneously increasing CD8+ T-cell immunogenicity after
administration to a chordate, the methods comprising the steps of:
1) identifying a B-cell epitope in a polypeptide; and 2) disrupting
the identified B-cell epitope with one or more amino acid
residue(s) comprised by a heterologous CD8+ T-cell epitope added to
the polypeptide. In certain further embodiments, the disrupting
step further comprises the creation of one or more amino acid
substitutions in the B-cell epitope region. In certain further
embodiments, the disrupting step further comprises creating one or
more amino acid insertions in the B-cell epitope region.
[0384] In certain embodiments of the methods of the present
invention, a CD4+ T-cell de-immunized polypeptide is created which
has reduced CD4+ T-cell immunogenicity when administered to a
chordate. In certain embodiments of the methods of the present
invention, is a method for reducing CD4+ T-cell immunogenicity in a
polypeptide, the method comprising the step of disrupting a CD4+
T-cell epitope region within a polypeptide with one or more amino
acid residue(s) comprised by a heterologous CD8+ T-cell epitope
added to the polypeptide. In certain further embodiments, the
disrupting step further comprises creating one or more amino acid
substitutions in the B-cell epitope region. In certain further
embodiments, the disrupting step further comprises creating one or
more amino acid insertions in the CD4+ T-cell epitope region.
[0385] Certain embodiments of the methods of the present invention
are methods for reducing CD4+ T-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity after administration to a chordate, the methods
comprising the step of disrupting a CD4+ T-cell epitope region
within a polypeptide with one or more amino acid residue(s)
comprised by a heterologous CD8+ T-cell epitope added to the
polypeptide. In certain further embodiments, the disrupting step
further comprises creating one or more amino acid substitutions in
the CD4+ T-cell epitope region. In certain further embodiments, the
disrupting step further comprises creating one or more amino acid
insertions in the CD4+ T-cell epitope region.
[0386] Certain embodiments of the methods of the present invention
are methods for reducing CD4+ T-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity after administration to a chordate, the methods
comprising the steps of: 1) identifying a CD4+ T-cell epitope in a
polypeptide; and 2) disrupting the identified CD4+ T-cell epitope
with one or more amino acid residue(s) comprised by a heterologous
CD8+ T-cell epitope added to the polypeptide. In certain further
embodiments, the disrupting step further comprises the creation of
one or more amino acid substitutions in the CD4+ T-cell epitope
region. In certain further embodiments, the disrupting step further
comprises creating one or more amino acid insertions in the CD4+
T-cell epitope region.
[0387] Certain embodiments of the methods of the present invention
are methods for reducing CD4+ T-cell immunogenicity in a
polypeptide while simultaneously increasing CD8+ T-cell
immunogenicity after administration to a chordate, the methods
comprising the steps of: 1) identifying a CD4+ T-cell epitope in a
polypeptide; and 2) disrupting the identified CD4+ T-cell epitope
with one or more amino acid residue(s) comprised by a heterologous
CD8+ T-cell epitope added to the polypeptide. In certain further
embodiments, the disrupting step further comprises the creation of
one or more amino acid substitutions in the CD4+ T-cell epitope
region. In certain further embodiments, the disrupting step further
comprises creating one or more amino acid insertions in the CD4+
T-cell epitope region.
XIII. Methods for Using a T-Cell Hyper-Immunized and/or B-Cell/CD4+
T-Cell De-Immunized Polypeptide of the Present Invention,
Cell-Targeted Molecule Comprising the Same, or Pharmaceutical
and/or Diagnostic Composition Thereof
[0388] Generally, it is an object of the invention to provide
pharmacologically active agents, as well as compositions comprising
the same, that can be used in the prevention and/or treatment of
diseases, disorders, and conditions, such as certain cancers,
tumors, growth abnormalities, immune disorders, or further
pathological conditions mentioned herein. Accordingly, the present
invention provides methods of using the polypeptides, cell-targeted
molecules, and pharmaceutical compositions of the present invention
for the delivering of T-cell epitopes to the MHC class I
presentation pathway of target cells, targeted killing of cells,
for delivering additional exogenous materials into targeted cells,
for labeling of the interiors of targeted cells, for collecting
diagnostic information, and for treating diseases, disorders, and
conditions as described herein.
[0389] Already cytotoxic molecules, such as e.g. potential
therapeutics comprising cytotoxic toxin region polypeptides, may be
engineered to be more cytotoxic and/or to have redundant, backup
cytotoxicities operating via completely different mechanisms. These
multiple cytotoxic mechanisms may complement each other (such as by
providing both two mechanisms of cell killing, direct and indirect,
as well as mechanisms of immuno-stimulation to the local area),
redundantly backup each other (such as by providing direct cell
killing in the absence of the other), and/or protect against
developed resistance (by limiting resistance to the less probable
situation of the malignant or infected cell blocking two different
mechanisms simultaneously).
[0390] In addition, parental cytotoxic molecules which rely on
toxin effector and/or enzymatic regions for cytotoxicity may be
engineered by mutating the parental molecule to be enzymatically
inactive but to be cytotoxic via T-cell epitope delivery to the MHC
class I system of a target cell and subsequent presentation to the
surface of the target cell. This approach removes one cytotoxic
mechanism while adding another and adds the capability of
immuno-stimulation to the local area of the target cell by T-cell
epitope presentation. Furthermore, parental cytotoxic molecules
which rely on enzymatic regions for cytotoxicity may be engineered
to be cytotoxic only via T-cell epitope delivery to the MHC class I
system by embedding a T-cell epitope in the enzymatic domain of the
parental molecule such that the enzymatic activity is reduced or
eliminated. This allows for the one-step modification of
enzymatically-cytotoxic molecules, which have the ability once in
an endosomal compartment to route to the cytosol and/or ER, into
enzymatically inactive, cytotoxic molecules which rely on T-cell
epitope delivery to the MHC class I system of a target cell and
subsequent presentation on the surface of the target cell for
cytotoxicity. Any of the polypeptides of the invention can be
engineered into cell-targeted cytotoxic molecules with potential as
therapeutics by the linking of a variety of cell-targeting binding
regions which target specific cell-type(s) within a mixture of two
or more cell types, such as, e.g., within an organism.
[0391] In particular, it is an object of the invention to provide
such pharmacologically active agents, compositions, and/or methods
that have certain advantages compared to the agents, compositions,
and/or methods that are currently known in the art. Accordingly,
the present invention provides methods of using polypeptides and
proteins with characterized by polypeptide sequences and
pharmaceutical compositions thereof. For example, any of the
polypeptide sequences in SEQ ID NOs: 1-60 may be specifically
utilized as a component of the cell-targeted molecules used in the
following methods.
[0392] The present invention provides methods of killing a cell
comprising the step of contacting the cell, either in vitro or in
vivo, with a polypeptide, protein, or pharmaceutical composition of
the present invention. The polypeptides, proteins, and
pharmaceutical compositions of the present invention can be used to
kill a specific cell type upon contacting a cell or cells with one
of the claimed compositions of matter. In certain embodiments, a
cytotoxic polypeptide, protein, or pharmaceutical composition of
the present invention can be used to kill specific cell types in a
mixture of different cell types, such as mixtures comprising cancer
cells, infected cells, and/or hematological cells. In certain
embodiments, a cytotoxic polypeptide, protein, or pharmaceutical
composition of the present invention can be used to kill cancer
cells in a mixture of different cell types. In certain embodiments,
a cytotoxic polypeptide, protein, or pharmaceutical composition of
the present invention can be used to kill specific cell types in a
mixture of different cell types, such as pre-transplantation
tissues. In certain embodiments, a polypeptide, protein, or
pharmaceutical composition of the present invention can be used to
kill specific cell types in a mixture of cell types, such as
pre-administration tissue material for therapeutic purposes. In
certain embodiments, a polypeptide, protein, or pharmaceutical
composition of the present invention can be used to selectively
kill cells infected by viruses or microorganisms, or otherwise
selectively kill cells expressing a particular extracellular target
biomolecule, such as a cell surface biomolecule. The polypeptides,
proteins, and pharmaceutical compositions of the present invention
have varied applications, including, e.g., uses in depleting
unwanted cell types from tissues either in vitro or in vivo, uses
in modulating immune responses to treat graft-versus-host disease,
uses as antiviral agents, uses as anti-parasitic agents, and uses
in purging transplantation tissues of unwanted cell types.
[0393] In certain embodiments, a cytotoxic polypeptide, protein, or
pharmaceutical composition of the present invention, alone or in
combination with other compounds or pharmaceutical compositions,
can show potent cell-kill activity when administered to a
population of cells, in vitro or in vivo in a subject such as in a
patient in need of treatment. By targeting the delivery of
enzymatically active toxin regions and T-cell epitopes using
high-affinity binding regions to specific cell types, this potent
cell-kill activity can be restricted to specifically and
selectively kill certain cell types within an organism, such as
certain cancer cells, neoplastic cells, malignant cells,
non-malignant tumor cells, or infected cells.
[0394] The present invention provides a method of killing a cell in
a patient in need thereof, the method comprising the step of
administering to the patient at least one cytotoxic polypeptide or
protein of the present invention, or a pharmaceutical composition
thereof.
[0395] Certain embodiments of the cytotoxic polypeptide, protein,
or pharmaceutical compositions thereof can be used to kill a cancer
cell in a patient by targeting an extracellular biomolecule found
physically coupled with a cancer or tumor cell. The terms "cancer
cell" or "cancerous cell" refers to various neoplastic cells which
grow and divide in an abnormally accelerated fashion and will be
clear to the skilled person. The term "tumor cell" includes both
malignant and non-malignant cells. Generally, cancers and/or tumors
can be defined as diseases, disorders, or conditions that are
amenable to treatment and/or prevention. The cancers and tumors
(either malignant or non-malignant) which are comprised of cancer
cells and/or tumor cells which may benefit from methods and
compositions of the invention will be clear to the skilled person.
Neoplastic cells are often associated with one or more of the
following: unregulated growth, lack of differentiation, local
tissue invasion, angiogenesis, and metastasis.
[0396] Certain embodiments of the cytotoxic polypeptide or
cell-targeted molecule of the present invention, or pharmaceutical
compositions thereof, can be used to kill an immune cell (whether
healthy or malignant) in a patient by targeting an extracellular
biomolecule found physically coupled with an immune cell.
[0397] Certain embodiments of the cytotoxic polypeptide or
cell-targeted molecule of the present invention, or pharmaceutical
compositions thereof, can be used to kill an infected cell in a
patient by targeting an extracellular biomolecule found physically
coupled with an infected cell.
[0398] It is within the scope of the present invention to utilize
the cell-targeted molecule of the present invention or
pharmaceutical composition thereof for the purposes of purging
patient cell populations (e.g. bone marrow) of malignant,
neoplastic, or otherwise unwanted T-cells and/or B-cells and then
reinfusing the T-cell and/or B-cells depleted material into the
patient (see e.g. van Heeckeren W et al., Br J Haematol 132: 42-55
(2006); (see e.g. Alpdogan O, van den Brink M, Semin Oncol 39:
629-42 (2012)).
[0399] It is within the scope of the present invention to utilize
the cell-targeted molecule of the present invention or
pharmaceutical composition thereof for the purposes of ex vivo
depletion of T cells and/or B-cells from isolated cell populations
removed from a patient. In one non-limiting example, the
cell-targeted molecule of the invention can be used in a method for
prophylaxis of organ and/or tissue transplant rejection wherein the
donor organ or tissue is perfused prior to transplant with a
cytotoxic, cell-targeted molecule of the invention or a
pharmaceutical composition thereof in order to purge the organ of
donor T-cells and/or B-cells (see e.g. Alpdogan O, van den Brink M,
Semin Oncol 39: 629-42 (2012)).
[0400] It is also within the scope of the present invention to
utilize the cell-targeted molecule of the invention or
pharmaceutical composition thereof for the purposes of depleting
T-cells and/or B-cells from a donor cell population as a
prophylaxis against graft-versus-host disease, and induction of
tolerance, in a patient to undergo a bone marrow and or stem cell
transplant (see e.g. van Heeckeren W et al., Br J Haematol 132:
42-55 (2006); (see e.g. Alpdogan O, van den Brink M, Semin Oncol
39: 629-42 (2012)).
[0401] Certain embodiments of the cytotoxic polypeptide or
cell-targeted molecule of the invention, or pharmaceutical
compositions thereof, can be used to kill an infected cell in a
patient by targeting an extracellular biomolecule found physically
coupled with an infected cell.
[0402] Certain embodiments of the cell-targeted molecules of the
present invention, or pharmaceutical compositions thereof, can be
used to "seed" a locus within an organism with non-self, T-cell
epitope-peptide presenting cells in order to activate the immune
system to police the locus. In certain further embodiments of this
"seeding" method of the present invention, the locus is a tumor
mass or infected tissue site. In preferred embodiments of this
"seeding" method of the present invention, the non-self, T-cell
epitope-peptide is selected from the group consisting of: peptides
not already presented by the target cells of the cell-targeted
molecule, peptides not present within any protein expressed by the
target cell, peptides not present within the proteome of the target
cell, peptides not present in the extracellular microenvironment of
the site to be seeded, and peptides not present in the tumor mass
or infect tissue site to be targeted.
[0403] This "seeding" method functions to label one or more target
cells within a chordate with one or more MHC class I presented
T-cell epitopes for recognition by effector T-cells and activation
of downstream immune responses. By exploiting the cell
internalizing, intracellularly routing, and T-cell epitope
delivering functions of the cell-targeted molecules of the
invention, the target cells which display the delivered T-cell
epitope are harnessed to induce recognition of the presenting
target cell by host T-cells and induction of further immune
responses including target cell killing by CTLs. This "seeding"
method of using a cell-targeted molecule of the present invention
can provide a temporary vaccination-effect by inducing adaptive
immune responses to attack the cells within the seeded
microenvironment, such as, e.g. a tumor mass or infected tissue
site, whether presenting a cell-targeted molecule-delivered T-cell
epitope(s) or not. This "seeding" method may also induce the
breaking of immuno-tolerance to a target cell population, a tumor
mass, and/or infected tissue site within an organism.
[0404] Additionally, the present invention provides a method of
treating a disease, disorder, or condition in a patient comprising
the step of administering to a patient in need thereof a
therapeutically effective amount of at least one of the cytotoxic
polypeptide or cell-targeted molecule of the present invention, or
a pharmaceutical composition thereof. Contemplated diseases,
disorders, and conditions that can be treated using this method
include cancers, malignant tumors, non-malignant tumors, growth
abnormalities, immune disorders, and microbial infections.
Administration of a "therapeutically effective dosage" of a
compound of the invention can result in a decrease in severity of
disease symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction.
[0405] The therapeutically effective amount of a compound of the
present invention will depend on the route of administration, the
type of mammal being treated, and the physical characteristics of
the specific patient under consideration. These factors and their
relationship to determining this amount are well known to skilled
practitioners in the medical arts. This amount and the method of
administration can be tailored to achieve optimal efficacy, and may
depend on such factors as weight, diet, concurrent medication and
other factors, well known to those skilled in the medical arts. The
dosage sizes and dosing regimen most appropriate for human use may
be guided by the results obtained by the present invention, and may
be confirmed in properly designed clinical trials. An effective
dosage and treatment protocol may be determined by conventional
means, starting with a low dose in laboratory animals and then
increasing the dosage while monitoring the effects, and
systematically varying the dosage regimen as well. Numerous factors
may be taken into consideration by a clinician when determining an
optimal dosage for a given subject. Such considerations are known
to the skilled person.
[0406] An acceptable route of administration may refer to any
administration pathway known in the art, including but not limited
to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal,
vaginal, or transdermal (e.g. topical administration of a cream,
gel or ointment, or by means of a transdermal patch). "Parenteral
administration" is typically associated with injection at or in
communication with the intended site of action, including
infraorbital, infusion, intraarterial, intracapsular, intracardiac,
intradermal, intramuscular, intraperitoneal, intrapulmonary,
intraspinal, intrastemal, intrathecal, intrauterine, intravenous,
subarachnoid, subcapsular, subcutaneous, transmucosal, or
transtracheal administration.
[0407] For administration of a pharmaceutical composition of the
present invention, the dosage range will generally be from about
0.0001 to 100 milligrams per kilogram (mg/kg), and more, usually
0.01 to 5 mg/kg, of the host body weight. Exemplary dosages may be
0.25 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5
mg/kg body weight or 10 mg/kg body weight or within the range of
1-10 mg/kg. An exemplary treatment regime is a once or twice daily
administration, or a once or twice weekly administration, once
every two weeks, once every three weeks, once every four weeks,
once a month, once every two or three months or once every three to
6 months. Dosages may be selected and readjusted by the skilled
health care professional as required to maximize therapeutic
benefit for a particular patient.
[0408] Pharmaceutical compositions of the present invention will
typically be administered to the same patient on multiple
occasions. Intervals between single dosages can be, for example,
2-5 days, weekly, monthly, every two or three months, every six
months, or yearly. Intervals between administrations can also be
irregular, based on regulating blood levels or other markers in the
subject or patient. Dosage regimens for a compound of the invention
include intravenous administration of 1 mg/kg body weight or 3
mg/kg body weight with the compound administered every two to four
weeks for six dosages, then every three months at 3 mg/kg body
weight or 1 mg/kg body weight.
[0409] A pharmaceutical composition of the present invention may be
administered via one or more routes of administration, using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled worker, the route and/or mode of
administration will vary depending upon the desired results. Routes
of administration for polypeptides, proteins, and pharmaceutical
compositions of the invention include, e.g. intravenous,
intramuscular, intradermal, intraperitoneal, subcutaneous, spinal,
or other parenteral routes of administration, for example by
injection or infusion. In other embodiments, a polypeptide,
protein, or pharmaceutical composition of the invention may be
administered by a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually, or
topically.
[0410] Therapeutic polypeptides, proteins, or pharmaceutical
compositions of the present invention may be administered with one
or more of a variety of medical devices known in the art. For
example, in one embodiment, a pharmaceutical composition of the
invention may be administered with a needleless hypodermic
injection device. Examples of well-known implants and modules
useful in the present invention are in the art, including e.g.,
implantable micro-infusion pumps for controlled rate delivery;
devices for administering through the skin; infusion pumps for
delivery at a precise infusion rate; variable flow implantable
infusion devices for continuous drug delivery; and osmotic drug
delivery systems. These and other such implants, delivery systems,
and modules are known to those skilled in the art.
[0411] A polypeptide, protein, or pharmaceutical composition of the
present invention may be administered alone or in combination with
one or more other therapeutic or diagnostic agents. A combination
therapy may include a cytotoxic, cell-targeted molecule of the
invention or pharmaceutical composition thereof combined with at
least one other therapeutic agent selected based on the particular
patient, disease or condition to be treated. Examples of other such
agents include, inter alia, a cytotoxic, anti-cancer or
chemotherapeutic agent, an anti-inflammatory or anti-proliferative
agent, an antimicrobial or antiviral agent, growth factors,
cytokines, an analgesic, a therapeutically active small molecule or
polypeptide, a single chain antibody, a classical antibody or
fragment thereof, or a nucleic acid molecule which modulates one or
more signaling pathways, and similar modulating therapeutics which
may complement or otherwise be beneficial in a therapeutic or
prophylactic treatment regimen.
[0412] Treatment of a patient with a polypeptide, protein, or
pharmaceutical composition of the present invention preferably
leads to cell death of targeted cells and/or the inhibition of
growth of targeted cells. As such, cytotoxic, cell-targeted
molecules of the present invention, and pharmaceutical compositions
comprising them, will be useful in methods for treating a variety
of pathological disorders in which killing or depleting target
cells may be beneficial, such as, inter alia, cancer, tumors, other
growth abnormalities, immune disorders, and infected cells. The
present invention provides methods for suppressing cell
proliferation, and treating cell disorders, including neoplasia,
overactive B-cells, and overactive T-cells.
[0413] In certain embodiments, polypeptides, proteins, and
pharmaceutical compositions of the present invention can be used to
treat or prevent cancers, tumors (malignant and non-malignant),
growth abnormalities, immune disorders, and microbial infections.
In a further aspect, the above ex vivo method can be combined with
the above in vivo method to provide methods of treating or
preventing rejection in bone marrow transplant recipients, and for
achieving immunological tolerance.
[0414] In certain embodiments, the present invention provides
methods for treating malignancies or neoplasms and other blood cell
associated cancers in a mammalian subject, such as a human, the
method comprising the step of administering to a subject in need
thereof a therapeutically effective amount of a cytotoxic protein
or pharmaceutical composition of the invention.
[0415] The cytotoxic polypeptides, proteins, and pharmaceutical
compositions of the present invention have varied applications,
including, e.g., uses in removing unwanted T-cells, uses in
modulating immune responses to treat graft-versus-host disease,
uses as antiviral agents, uses as antimicrobial agents, and uses in
purging transplantation tissues of unwanted cell types. The
cytotoxic polypeptides, proteins, and pharmaceutical compositions
of the present invention are commonly anti-neoplastic
agents--meaning they are capable of treating and/or preventing the
development, maturation, or spread of neoplastic or malignant cells
by inhibiting the growth and/or causing the death of cancer or
tumor cells.
[0416] In certain embodiments, a polypeptide, protein, or
pharmaceutical composition of the present invention is used to
treat a B-cell-, plasma cell- or antibody-mediated disease or
disorder, such as for example leukemia, lymphoma, myeloma, Human
Immunodeficiency Virus-related diseases, amyloidosis, hemolytic
uremic syndrome, polyarteritis, septic shock, Crohn's Disease,
rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis,
ulcerative colitis, psoriasis, asthma, Sjorgren's syndrome,
graft-versus-host disease, graft rejection, diabetes, vasculitis,
scleroderma, and systemic lupus erythematosus.
[0417] In another aspect, certain embodiments of the polypeptides,
proteins, and pharmaceutical compositions of the present invention
are antimicrobial agents--meaning they are capable of treating
and/or preventing the acquisition, development, or consequences of
microbiological pathogenic infections, such as caused by viruses,
bacteria, fungi, prions, or protozoans.
[0418] It is within the scope of the present invention to provide a
prophylaxis or treatment for diseases or conditions mediated by
T-cells or B-cells by administering the cytotoxic protein or the
invention, or a pharmaceutical composition thereof, to a patient
for the purpose of killing T-cells or B-cells in the patient. This
usage is compatible with preparing or conditioning a patient for
bone marrow transplantation, stem cell transplantation, tissue
transplantation, or organ transplantation, regardless of the source
of the transplanted material, e.g. human or non-human sources.
[0419] It is within the scope of the present invention to provide a
bone marrow recipient for prophylaxis or treatment of
host-versus-graft disease via the targeted cell-killing of host
T-cells using a cytotoxic polypeptide, protein, or pharmaceutical
composition of the present invention.
[0420] The cytotoxic polypeptides, proteins, and pharmaceutical
compositions of the present invention can be utilized in a method
of treating cancer comprising administering to a patient, in need
thereof, a therapeutically effective amount of a cytotoxic
polypeptide, protein, or pharmaceutical composition of the present
invention. In certain embodiments of the methods of the present
invention, the cancer being treated is selected from the group
consisting of: bone cancer (such as multiple myeloma or Ewing's
sarcoma), breast cancer, central/peripheral nervous system cancer
(such as brain cancer, neurofibromatosis, or glioblastoma),
gastrointestinal cancer (such as stomach cancer or colorectal
cancer), germ cell cancer (such as ovarian cancers and testicular
cancers, glandular cancer (such as pancreatic cancer, parathyroid
cancer, pheochromocytoma, salivary gland cancer, or thyroid
cancer), head-neck cancer (such as nasopharyngeal cancer, oral
cancer, or pharyngeal cancer), hematological cancers (such as
leukemia, lymphoma, or myeloma), kidney-urinary tract cancer (such
as renal cancer and bladder cancer), liver cancer, lung/pleura
cancer (such as mesothelioma, small cell lung carcinoma, or
non-small cell lung carcinoma), prostate cancer, sarcoma (such as
angiosarcoma, fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma),
skin cancer (such as basal cell carcinoma, squamous cell carcinoma,
or melanoma), and uterine cancer.
[0421] The polypeptides, proteins, and pharmaceutical compositions
of the present invention can be utilized in a method of treating an
immune disorder comprising administering to a patient, in need
thereof, a therapeutically effective amount of the cytotoxic
protein or a pharmaceutical composition of the present invention.
In certain embodiments of the methods of the present invention, the
immune disorder is related to an inflammation associated with a
disease selected from the group consisting of: amyloidosis,
ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft
rejection, graft-versus-host disease, Hashimoto's thyroiditis,
hemolytic uremic syndrome, HIV-related diseases, lupus
erythematosus, multiple sclerosis, polyarteritis, psoriasis,
psoriatic arthritis, rheumatoid arthritis, scleroderma, septic
shock, Sjorgren's syndrome, ulcerative colitis, and vasculitis.
[0422] Among certain embodiments of the present invention is using
the polypeptide or cell-targeted molecule of the present invention
as a component of a pharmaceutical composition or medicament for
the treatment or prevention of a cancer, tumor, other growth
abnormality, immune disorder, and/or microbial infection. For
example, immune disorders presenting on the skin of a patient may
be treated with such a medicament in efforts to reduce
inflammation. In another example, skin tumors may be treated with
such a medicament in efforts to reduce tumor size or eliminate the
tumor completely.
[0423] Certain cytotoxic polypeptides, proteins, and pharmaceutical
compositions of the present invention may be used in molecular
neurosurgery applications such as immunolesioning and neuronal
tracing (see, Wiley R, Lappi D, Adv Drug Deliv Rev 55: 1043-54
(2003), for review). For example, the targeting domain may be
selected or derived from various ligands, such as neurotransmitters
and neuropeptides, which target specific neuronal cell types by
binding neuronal surface receptors, such as a neuronal circuit
specific G-protein coupled receptor. Similarly, the targeting
domain may be selected from or derived from antibodies that bind
neuronal surface receptors. Because certain toxins robustly direct
their own retrograde axonal transport, certain cytotoxic,
cell-targeted molecules of the invention may be used to kill a
neuron(s) which expresses the extracellular target at a site of
cytotoxic protein injection distant from the cell body (see
Llewellyn-Smith I et al., J Neurosci Methods 103: 83-90 (2000)).
These neuronal cell type specific targeting cytotoxic polypeptides
and proteins have uses in neuroscience research, such as for
elucidating mechanisms of sensations (see e.g. Mishra S, Hoon M,
Science 340: 968-71 (2013), and creating model systems of
neurodegenerative diseases, such as Parkinson's and Alzheimer's
(see e.g. Hamlin A et al., PLoS One e53472 (2013)).
[0424] Among certain embodiment of the present invention is a
method of using a polypeptide, protein, pharmaceutical composition,
and/or diagnostic composition of the present invention to label or
detect the interiors of neoplastic cells and/or immune cell types.
Based on the ability of certain polypeptides, proteins, and
pharmaceutical compositions of the invention to enter specific cell
types and route within cells via retrograde intracellular
transport, the interior compartments of specific cell types are
labeled for detection. This can be performed on cells in situ
within a patient or on cells and tissues removed from an organism,
e.g. biopsy material.
[0425] Among certain embodiment of the present invention is a
method of using a polypeptide, protein, pharmaceutical composition,
and/or diagnostic composition of the present invention to detect
the presence of a cell type for the purpose of information
gathering regarding diseases, conditions and/or disorders. The
method comprises contacting a cell with a diagnostically sufficient
amount of a cytotoxic molecule to detect the cytotoxic molecule by
an assay or diagnostic technique. The phrase "diagnostically
sufficient amount" refers to an amount that provides adequate
detection and accurate measurement for information gathering
purposes by the particular assay or diagnostic technique utilized.
Generally, the diagnostically sufficient amount for whole organism
in vivo diagnostic use will be a non-cumulative dose of between 0.1
mg to 100 mg of the detection promoting agent linked cell-targeted
molecule of the invention per kg of subject per subject. Typically,
the amount of polypeptide or cell-targeted molecule of the
invention used in these information gathering methods will be as
low as possible provided that it is still a diagnostically
sufficient amount. For example, for in vivo detection in an
organism, the amount of polypeptide, protein, or pharmaceutical
composition of the invention administered to a subject will be as
low as feasibly possible.
[0426] The cell-type specific targeting of polypeptides and
cell-targeted molecules of the present invention combined with
detection promoting agents provides a way to detect and image cells
physically coupled with an extracellular target biomolecule of a
binding region of the molecule of the invention. Imaging of cells
using the polypeptides or cell-targeted molecules of the present
invention may be performed in vitro or in vivo by any suitable
technique known in the art. Diagnostic information may be collected
using various methods known in the art, including whole body
imaging of an organism or using ex vivo samples taken from an
organism. The term "sample" used herein refers to any number of
things, but not limited to, fluids such as blood, urine, serum,
lymph, saliva, anal secretions, vaginal secretions, and semen, and
tissues obtained by biopsy procedures. For example, various
detection promoting agents may be utilized for non-invasive in vivo
tumor imaging by techniques such as magnetic resonance imaging
(MRI), optical methods (such as direct, fluorescent, and
bioluminescent imaging), positron emission tomography (PET),
single-photon emission computed tomography (SPECT), ultrasound,
x-ray computed tomography, and combinations of the aforementioned
(see, Kaur S et al., Cancer Lett 315: 97-111 (2012), for
review).
[0427] Among certain embodiment of the present invention is a
method of using a polypeptide, protein, or pharmaceutical
composition of the present invention as a diagnostic composition to
label or detect the interiors of cancer, tumor, and/or immune cell
types (see e.g., Koyama Y et al., Clin Cancer Res 13: 2936-45
(2007); Ogawa M et al., Cancer Res 69: 1268-72 (2009); Yang L et
al., Small 5: 235-43 (2009)). Based on the ability of certain
polypeptides, proteins, and pharmaceutical compositions of the
invention to enter specific cell types and route within cells via
retrograde intracellular transport, the interior compartments of
specific cell types are labeled for detection. This can be
performed on cells in situ within a patient or on cells and tissues
removed from an organism, e.g. biopsy material.
[0428] Diagnostic compositions of the present invention may be used
to characterize a disease, disorder, or condition as potentially
treatable by a related pharmaceutical composition of the present
invention. Certain compositions of matter of the present invention
may be used to determine whether a patient belongs to a group that
responds to a therapeutic strategy which makes use of a compound,
composition or related method of the present invention as described
herein or is well suited for using a delivery device of the
invention.
[0429] Diagnostic compositions of the present invention may be used
after a disease, e.g. a cancer, is detected in order to better
characterize it, such as to monitor distant metastases,
heterogeneity, and stage of cancer progression. The phenotypic
assessment of disease disorder or infection can help prognostic and
prediction during therapeutic decision making. In disease
reoccurrence, certain methods of the invention may be used to
determine if local or systemic problem.
[0430] Diagnostic compositions of the present invention may be used
to assess responses to therapeutic(s) regardless of the type of
therapeutic, e.g. small molecule drug, biological drug, or
cell-based therapy. For example, certain embodiments of the
diagnostics of the invention may be used to measure changes in
tumor size, changes in antigen positive cell populations including
number and distribution, or monitoring a different marker than the
antigen targeted by a therapy already being administered to a
patient (see Smith-Jones P et al., Nat. Biotechnol 22: 701-6
(2004); Evans M et al., Proc. Natl. Acad. Sci. U.S.A. 108: 9578-82
(2011)).
[0431] Certain embodiments of the method used to detect the
presence of a cell type may be used to gather information regarding
diseases, disorders, and conditions, such as, for example bone
cancer (such as multiple myeloma or Ewing's sarcoma), breast
cancer, central/peripheral nervous system cancer (such as brain
cancer, neurofibromatosis, or glioblastoma), gastrointestinal
cancer (such as stomach cancer or colorectal cancer), germ cell
cancer (such as ovarian cancers and testicular cancers, glandular
cancer (such as pancreatic cancer, parathyroid cancer,
pheochromocytoma, salivary gland cancer, or thyroid cancer),
head-neck cancer (such as nasopharyngeal cancer, oral cancer, or
pharyngeal cancer), hematological cancers (such as leukemia,
lymphoma, or myeloma), kidney-urinary tract cancer (such as renal
cancer and bladder cancer), liver cancer, lung/pleura cancer (such
as mesothelioma, small cell lung carcinoma, or non-small cell lung
carcinoma), prostate cancer, sarcoma (such as angiosarcoma,
fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer
(such as basal cell carcinoma, squamous cell carcinoma, or
melanoma), uterine cancer, AIDS, amyloidosis, ankylosing
spondylitis, asthma, autism, cardiogenesis, Crohn's disease,
diabetes, erythematosus, gastritis, graft rejection,
graft-versus-host disease, Grave's disease, Hashimoto's
thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus
erythematosus, lymphoproliferative disorders, multiple sclerosis,
myasthenia gravis, neuroinflammation, polyarteritis, psoriasis,
psoriatic arthritis, rheumatoid arthritis, scleroderma, septic
shock, Sjorgren's syndrome, systemic lupus erythematosus,
ulcerative colitis, vasculitis, cell proliferation, inflammation,
leukocyte activation, leukocyte adhesion, leukocyte chemotaxis,
leukocyte maturation, leukocyte migration, neuronal
differentiation, acute lymphoblastic leukemia (ALL), T acute
lymphocytic leukemia/lymphoma (ALL), acute myelogenous leukemia,
acute myeloid leukemia (AML), B-cell chronic lymphocytic leukemia
(B-CLL), B-cell prolymphocytic lymphoma, Burkitt's lymphoma (BL),
chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia
(CML-BP), chronic myeloid leukemia (CML), diffuse large B-cell
lymphoma, follicular lymphoma, hairy cell leukemia (HCL), Hodgkin's
Lymphoma (HL), intravascular large B-cell lymphoma, lymphomatoid
granulomatosis, lymphoplasmacytic lymphoma, MALT lymphoma, mantle
cell lymphoma, multiple myeloma (MM), natural killer cell leukemia,
nodal marginal B-cell lymphoma, Non-Hodgkin's lymphoma (NHL),
plasma cell leukemia, plasmacytoma, primary effusion lymphoma,
pro-lymphocytic leukemia, promyelocytic leukemia, small lymphocytic
lymphoma, splenic marginal zone lymphoma, T-cell lymphoma (TCL),
heavy chain disease, monoclonal gammopathy, monoclonal
immunoglobulin deposition disease, myelodusplastic syndromes (MDS),
smoldering multiple myeloma, and Waldenstrom macroglobulinemia.
[0432] In certain embodiments, the polypeptides and cell-targeted
molecules of the present invention, or pharmaceutical compositions
thereof, are used for both diagnosis and treatment, or for
diagnosis alone. In some situations, it would be desirable to
determine or verify the HLA variant(s) and/or HLA alleles expressed
in the subject and/or diseased tissue from the subject, such as,
e.g., a patient in need of treatment, before selecting a
polypeptide or cell-targeted molecule of the invention for
treatment.
[0433] The present invention is further illustrated by the
following non-limiting examples of 1) CD8+ T-cell hyper-immunized
and/or B-cell/CD4+ T-cell de-immunized polypeptides, 2) CD8+ T-cell
epitope presenting toxin-derived polypeptides, and 3) selectively
cytotoxic, cell-targeted proteins comprising the aforementioned
polypeptides and capable of specifically targeting certain cell
types.
EXAMPLES
[0434] The following examples demonstrate certain embodiments of
the present invention. However, it is to be understood that these
examples are for illustration purposes only and do not intend, nor
should any be construed, to be wholly definitive as to conditions
and scope of this invention. The experiments in the following
examples were carried out using standard techniques, which are well
known and routine to those of skill in the art, except where
otherwise described in detail.
[0435] The presentation of a T-cell immunogenic epitope peptide by
the MHC class I system targets the presenting cell for killing by
CTL-mediated lysis and also triggers immune stimulation in the
local microenvironment. By engineering immunogenic epitope
sequences within toxin effector polypeptide components of
target-cell-internalizing therapeutics, the targeted delivery and
presentation of immuno-stimulatory antigens may be accomplished.
The presentation of immuno-stimulatory non-self antigens, such as
e.g. known viral antigens with high immunogenicity, by target cells
signals to other immune cells to destroy the target cells as well
as to recruit more immune cells to the area.
[0436] In the examples, T-cell epitopes were embedded or inserted
into Shiga toxin effector polypeptides and diphtheria toxin
effector polypeptides, which may serve as components of
target-cell-internalizing molecules, by engineering internal
regions to comprise one or more T-cell epitopes. Thus, there is no
terminal fusion of an additional amino acid residue, peptide, or
polypeptide component to the starting polypeptide.
[0437] In the examples, most of the T-cell epitopes were embedded
into toxin effector polypeptide components of
target-cell-internalizing molecules by engineering multiple amino
acid substitutions but without changing the total number of amino
acid residues in the exemplary toxin effector polypeptides as
compared to the parental toxin effector polypeptide. Thus, for all
of the diphtheria toxin effector polypeptides and most of the Shiga
toxin effector polypeptides tested in the Examples, there was no
insertion of additional amino acids but rather only substitutions
for existing amino acids resulting in the maintenance of the
original length of the parental polypeptide.
[0438] Novel toxin-derived effector polypeptides, which can
function as components of cell-targeted molecules (such as e.g.
immunotoxins and ligand-toxin fusions), were created which can
promote cellular internalization, sub-cellular routing to the
cytosol, and delivery of the T-cell epitope to the cytosol for
presentation by the MHC I class pathway to the target cell surface
to signal to CTLs.
[0439] Certain novel toxin-derived effector polypeptides were also
de-immunized by embedding or inserting a T-cell epitope in a B-cell
epitope region using one or more methods of the present invention.
In order to simultaneously de-immunize and provide for T-cell
epitope presentation on the target cell surface within the same
toxin polypeptide region, predicted B-cell epitope regions were
disrupted by replacing them with known T-cell epitopes predicted to
bind to MHC Class I molecules. Amino acid sequences from
toxin-derived polypeptides were analyzed to predict antigenic
and/or immunogenic B-cell epitopes in silico. Various T-cell
epitope embedded, toxin-derived polypeptides were experimentally
tested for retention of toxin effector functions.
[0440] The preservation of toxin effector functions of exemplary
T-cell epitope presenting toxin effector polypeptides of the
invention were tested and compared to toxin effector polypeptides
comprising wild-type toxin polypeptide sequences, referred to
herein as "wild-type" or "WT," which did not comprise any internal
modification or mutation to the toxin effector region.
[0441] The following examples of exemplary CD8+ T-cell epitope
presenting Shiga toxin-derived polypeptides of the invention
demonstrate methods of simultaneously providing for T-cell epitope
delivery for MHC class I presentation while retaining one or more
Shiga toxin effector functions. Further, the following examples of
exemplary CD8+ T-cell epitope presenting and/or B-cell/CD4+ T-cell
de-immunized Shiga toxin-derived polypeptides of the invention
demonstrate methods of simultaneously providing for 1) T-cell
epitope delivery for MHC class I presentation, 2) retaining one or
more toxin effector functions, and 3) de-immunization of the toxin
effector region.
[0442] The exemplary cell-targeted molecules of the invention bound
to target biomolecules expressed by targeted cell types and entered
the targeted cells. The internalized exemplary cell-targeted
proteins of the invention effectively routed their de-immunized
toxin effector regions to the cytosol and effectively delivered
immunogenic T-cell epitopes to the target cells' MHC class I
pathway resulting in presentation of the T-cell epitope peptide on
the surface of target cells regions.
Example 1
Embedding or Inserting T-Cell Epitopes within Polypeptide
Components of Cell-Targeting Molecules
[0443] In this example, T-cell epitope sequences were selected from
human viral proteins and embedded or inserted into Shiga toxin
effector polypeptides. In some variants, the T-cell epitope was
embedded or inserted into B-cell epitope regions in order to
disrupt natively occurring B-cell epitopes. In other variants, the
T-cell epitope is embedded into regions not predicted to contain
any B-cell epitopes and, thus, these modifications are not
predicted to disrupt any dominant B-cell epitopes. In some of the
above variants, the T-cell epitope is embedded into regions
predicted to disrupt catalytic activity.
A. Selecting T-Cell Epitope Peptides for Embedding or Insertion
[0444] In this example, known T-cell epitope peptides were selected
for embedding and inserting into Shiga toxin effector regions which
have the intrinsic ability to intracellularly route to the cytosol.
For example, there are many known immunogenic viral proteins and
peptide components of viral proteins from human viruses, such as
human influenza A viruses and human CMV viruses. Immunogenic viral
peptides were chosen that are capable of binding to human MHC class
I molecules and/or eliciting human CTL-mediated responses.
[0445] Seven peptides predicted to be T-cell epitopes (SEQ ID
NOs:4-10) were scored for the ability to bind to common human MHC
class I human leukocyte antigen (HLA) variants encoded by the more
prevalent alleles in human populations using the Immune Epitope
Database (IEDB) Analysis Resource MHC-I binding prediction's
consensus tool and recommended parameters (Kim Y et al., Nucleic
Acids Res 40: W252-30 (2012)). The IEDB MHC-I binding prediction
analysis predicted the "ANN affinity"--an estimated binding
affinity between the input peptide and the selected human HLA
variant where IC.sub.50 values less than 50 nanomolar (nM) are
considered high affinity, IC.sub.50 values between 50 and 500 nM
are considered intermediate affinity, and IC.sub.50 values between
500 and 5000 nM are considered low affinity. The IEDB MHC-I binding
prediction analysis indicated the best binders by the lowest
percentile ranks. Table 1 shows the IEDB MHC-I binding prediction
percentile rank and predicted binding affinity of the seven tested
T cell epitope-peptides (SEQ ID NOs:4-10) binding to the selected
human HLA variants.
TABLE-US-00004 TABLE 1 Predictions for Various Viral
Protein-Derived T-Cell Epitopes Binding to Various Human MHC Class
I Complexes IEDB MHC-I binding prediction T-cell epitope Percentile
Predicted Name Sequence HLA Allele Rank Affinity F2 GILGFVFTL
HLA-A*32:01 0.80 intermediate HLA-A*02:01 0.80 high HLA-A*02:06
2.20 high F2-2 DILGFVFTL HLA-A*32:01 1.40 intermediate HLA-A*02:01
4.60 low HLA-A*02:06 9.55 intermediate F2-3 DILGFDFTL HLA-A*32:01
2.80 low HLA-A*02:01 8.20 low HLA-A*02:06 11.25 low F2-4 GILGDVFTL
HLA-A*02:01 1.40 high HLA-A*02:06 2.40 high HLA-A*32:01 3.10 low F3
ILRGSVAHK HLA-A*03:01 0.25 high HLA-A*30:01 0.70 high HLA-A*31:01
4.25 intermediate F3-4 ILRFSVAHK HLA-A*03:01 0.25 high HLA-A*30:01
0.80 high HLA-A*31:01 3.30 intermediate C2 NLVPMVATV HLA-A*02:03
0.30 high HLA-A*02:01 1.00 high HLA-A*02:06 1.10 high
[0446] The results of the IEDB MHC-I binding prediction analysis
show that some peptides were predicted to bindwith high affinity to
multiple human MHC class I molecules, whereas other peptides were
predicted to bind with more moderate affinities to the analyzed
human MHC class I molecules.
B. Identifying B-Cell Epitope Regions in Toxins and Toxin Effector
Polypeptides
[0447] Toxin derived polypeptides with intrinsic subcellular
routing characteristics suitable for proteasome delivery were
chosen for de-immunization in order to reduce the possibility of
undesirable immune responses after administration to chordate, such
as, e.g., the production of anti-toxin antibodies. Amino acid
sequences from toxins and toxin-derived polypeptides were analyzed
to predict antigenic and/or immunogenic B-cell epitopes in
silico.
[0448] Polypeptide effectors derived from both a Shiga toxin and a
diphtheria toxin were analyzed for B-cell epitopes.
Shiga Toxin Derived Effector Polypeptides
[0449] First, B-cell epitope regions were identified within Shiga
toxin A Subunits. Computational methods were utilized to predict
antigenic and/or immunogenic B-cell epitopes in Shiga toxin A
subunit sequences with the potential to elicit responses by
mammalian immune systems after administration.
[0450] Linear B-cell epitopes were predicted within the A Subunits
of Shiga toxins using the polypeptide sequence and 3D structural
data of Shiga-Like Toxin Chain A (PDB ID: 1DM0_A) and the
REVEAL.RTM. system provided by ProImmune, Inc. (Sarasota, Fla.,
U.S.). In parallel, B-cell epitopes were predicted within the amino
acid sequences of the A Subunit of Shiga toxins using the BcePred
webserver (Saha S, Raghava G, Lecture Notes in Comput Sci 3239:
197-204 (2004)), Bepipred Linear Epitope Prediction (Larsen J et
al., Immunome Res 2: 2 (2006)), ElliPro Antibody epitope prediction
(Haste Andersen P et al., Protein Sci 15: 2558-67 (2006);
Ponomarenko J, Bourne P, BMC Struct Biol 7: 64 (2007)), and/or the
Epitopia server (Rubinstein N et al., BMC Bioinformatics 10: 287
(2009)). The Epitopia server prediction was used to identify
immunogenic B-cell epitopes as any stretch of linear amino acid
residues comprising a majority of residues predicted on Epitopia's
immunogenicity scale to be "high" (scored as 4 or 5). The various
computational methods revealed similar predictions for B-cell
epitope regions in the three prototypical Shiga toxin A Subunits
(Tables 2-4).
TABLE-US-00005 TABLE 2 B-Cell Epitope Predictions for the Mature,
Native A Subunit of Shiga-like Toxin 1 (SEQ ID NO: 1) Epitope
natively positioned amino acid positions Region REVEAL BcePred
Bepipred ElliPro Epitopia 1 1-15 2 29-35 28-34 27-37 18-23 3 42-48
39-46 43-47 44-49 4 58-66 55-61 56-64 57-66 52-62 5 96-103 105-111
100-115 96-110 94-102, 109- 114 6 144-151 141-147 147-151 144- 153
7 183-189 181-187 183-185 180- 179-188 190 8 211-219 211-220 9
243-251 243- 245-259 257 10 257-268 261-267 254-268 11 289-293
285-291 262- 262-281 293
TABLE-US-00006 TABLE 3 B-Cell Epitope Predictions for the Mature,
Native A Subunit of Shiga Toxin (SEQ ID NO: 2) natively positioned
amino acid positions REVEAL BcePred Bepipred ElliPro 29-35 28-34
27-37 42-48 39-46 44-47 58-66 55-61 56-64 57-66 96-103 105-111
100-115 96-110 144-151 141-147 147-151 144-153 183-189 181-187
183-185 180-190 211-219 243-251 243-257 257-268 261-267 254-268
289-293 285-291 262-293
TABLE-US-00007 TABLE 4 B-Cell Epitope Predictions for the Mature,
Native A Subunit of Shiga-like Toxin 2 (SEQ ID NO: 3) natively
positioned amino acid positions BcePred Bepipred ElliPro 3-11 8-14
29-35 28-36 26-37 42-48 57-62 56-66 108-115 109-115 96-110 141-156
140-153 179-188 180-191 210-218 210-217 240-257 244-258 241-255
262-278 281-297
[0451] In addition to Shiga toxin-derived toxin effector
polypeptides, which are capable of inducing cellular
internalization and directing their own subcellular routing to the
cytosol, cytosolic routing effector regions from other proteins may
be chosen as a source for polypeptides to modify into a polypeptide
of the present invention, such as, e.g., from other ABx and/or RIP
toxins.
Diphtheria Toxin Derived Effector Polypeptides
[0452] Diphtheria toxins have been used to design immunotoxins and
ligand-toxin fusion molecules wherein the diphtheria derived
component can provide cellular internalization and cytosolic
routing effector functions. A computational method was utilized to
predict antigenic and/or immunogenic B-cell epitope regions in the
diphtheria toxin A subunit with the potential to elicit responses
in mammalian immune systems. B-cell epitope regions were predicted
in the A Subunit of diphtheria toxin (SEQ ID NO:44) using the
BcePred webserver (Saha S, Raghava G, Lecture Notes in Comput Sci
3239: 197-204 (2004)). This computational method revealed seven
putative B-cell epitope regions in the prototypical Diphtheria
Toxin A Subunit (Table 5). In addition, the Immune Epitope Database
(IEDB) curated by the National Institutes of Allergy and Infectious
Diseases of the U.S. (NIAID) is said to provide all experimentally
characterized B-cell nd T-cell epitopes of diphtheria toxins.
Currently, the IEDB provides 7 epitopes with at least one positive
measurement regarding peptidic epitopes related to the diphtheria
toxin A subunit and the diphtheria toxin effector polypeptide SEQ
ID NO:44 used in the Examples (see Table 5 and region 182-201 and
225-238 of SEQ ID NO:44).
TABLE-US-00008 TABLE 5 B-Cell Epitope Predictions for the Mature,
Native A Subunit of Diphtheria Toxin (SEQ ID NO: 44) Natively
positioned amino acids Epitope Region BcePred IEDB 1 3-10 2 15-31 3
33-43 32-54 4 71-77 5 93-113 6 125-131 7 138-146 141-167 8 165-175
141-167 9 185-191 181-193
C. Identifying CD4+ T-Cell Epitope Regions in Toxins and Toxin
Effector Polypeptides
[0453] The Shiga toxin A subunit was analyzed for the presence of
any CD4+ T-cell epitopes. T-cell epitopes were predicted for the
mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) by the
REVEAL.TM. Immunogenicity System (IS) T-cell assay performed by
ProImmune Inc. (Sarasota, Fla., U.S.). This assay uses multiple
overlapping peptide sequences from the subject protein to test for
the elicitation of any immune response by CD4+ T-cells from healthy
donor cell samples depleted of CD8+ T-cells. There were seven
T-cell epitope regions identified using this assay at the following
natively positioned groups of amino acid residues: CD4+ T-cell
epitope region #1: 4-33, CD4+ T-cell epitope region #2: 34-78, CD4+
T-cell epitope region #3: 77-103, CD4+ T-cell epitope region #4:
128-168, CD4+ T-cell epitope region #5: 160-183, CD4+ T-cell
epitope region #6: 236-258, and CD4+ T-cell epitope region #7:
274-293.
[0454] The diphtheria toxin A subunit and a wild-type (WT),
diphtheria toxin effector polypeptide used as a parental
polypeptide for generation of the diphtheria toxin effector
polypeptides in the Examples, were investigated on NIAD's IEDB for
T-cell epitopes. Currently, the IEDB provides over 25 peptidic
epitopes with at least one positive measurement regarding T-cell
immunogenic related to the diphtheria toxin A subunit and the
diphtheria toxin effector polypeptides in the Examples. There were
several T-cell epitope regions identified by the IEDB in diphtheria
toxins, such as, e.g., the following regions corresponding to
overlapping immunogenic peptides in the polypeptide of SEQ ID NO:45
at amino acid residue positions: 2-21, 22-41, 32-71, 72-91, 82-221,
212-231, 232-251, and 251-301.
D. Generating Toxin Effector Polypeptides with Embedded or Inserted
T-Cell Epitopes Disrupting Endogenous B-Cell Epitope Regions and/or
Endogenous CD4+ T-Cell Epitope Regions
[0455] Exemplary toxin-derived effector polypeptides of the
invention were created using both a Shiga toxin and a diphtheria
toxin.
Shiga Toxin Derived Effector Polypeptides
[0456] A nucleic acid encoding a cytotoxic protein comprising a
Shiga toxin effector region and an immunoglobulin-type binding
region for cell targeting was produced using techniques known in
the art. The Shiga toxin effector region in the parental cytotoxic
protein of this example comprised amino acids 1-251 of SEQ ID
NO:1.
[0457] Using standard techniques known in the art, a series of
mutations were engineered into the nucleic acid encoding the
parental cytotoxic protein and variants of the cytotoxic protein
were produced which comprised multiple amino acid substitutions as
compared to the parental cytotoxic protein. The mutations were
selected to disrupt at least one predicted B-cell epitope region
described in Table 2 by embedding at least one T-cell epitope
peptide described in Table 1. For most of the exemplary
polypeptides of the invention described in the Examples, the amino
acid sequence for each T-cell epitope was embedded by manipulating
the nucleic acid sequences encoding the region of interest such
that the total number of encoded amino acid residues in the
variants remained unchanged from the total number of amino acid
residues in the parental cytotoxic protein. Ten different
polynucleotides were generated which each encoded a different
exemplary cytotoxic, cell-targeted protein of the invention
comprising a different exemplary Shiga toxin effector polypeptide
component of the invention. These exemplary polynucleotides were
used to produce ten exemplary cytotoxic, cell-targeted proteins of
the invention using standard techniques known in the art. In
certain experiments, the full-length coding sequence of the
cytotoxic protein of this example began or ended with a
polynucleotide encoding a Strep-tag.RTM. II to facilitate detection
and purification. Proteins were purified using methods known to the
skilled worker.
[0458] Eleven cytotoxic proteins were derived from the parental
cytotoxic protein, each comprising an exemplary Shiga toxin
effector polypeptide of the invention (selected from SEQ ID NOs:
11-21) and a disruption of at least one of the predicted B-cell
epitope regions in Table 2 using one of the T-cell epitopes
described in Table 1. The exact modification to the parental Shiga
toxin effector polypeptide for each of the eleven cytotoxic
proteins is shown in Table 6. Table 6 lists the sequence of each
embedded T-cell epitope, the native position in the Shiga toxin A
Subunit of the modification, and the disrupted stretch of amino
acids in the B-cell epitope region.
TABLE-US-00009 TABLE 6 Exemplary Shiga Toxin Effector Poly peptides
with T-Cell Epitopes Embedded or Inserted into B-Cell Epitope
Regions and/or CD4+ T-Cell Epitope Regions Position T-Cell B-Cell
(native residue Epitope T-Cell Epitope Epitope B-Cell Epitope
positions) Name Embedded Region Region Replaced 4-12 F3-4 ILRFSVAHK
1 TLDFSTAKT 43-51 C2 NLVPMVATV 3 SGSGDNLFA 44-52 F2 GILGFVFTL 3
GSGDNLFAV 44-52 C2 NLVPMVATV 3 GSGDNLFAV 53-61 F2-2 DILGFVFTL 4
DVRGIDPEE 53-61 F2-3 DILGFDFTL 4 DVRGIDPEE 53-61 C2 NLVPMVATV 4
DVRGIDPEE 104-112 C2 NLVPMVATV 5 TAVTLSGDS 180-188 F2-4 GILGDVFTL 7
TTLDDLSGR 53-61 F2 GILGFVFTL 4 DVRGIDPE 245/246 F3 ILRGSVAHK 9 none
(inserted at 246)
[0459] The first nine cytotoxic proteins each comprised a Shiga
toxin effector polypeptide comprising an embedded T-cell epitope
(see Table 6)--meaning without any change to the overall total
number of amino acid residues in the Shiga toxin effector
polypeptide component of the parental cytotoxic protein. Each of
the first nine modifications listed in Table 6 exemplifies an
embedded T-cell epitope which disrupts a B-cell epitope region. As
these nine modifications are exact replacements, the T-cell epitope
sequence and B-cell epitope region sequence disrupted are identical
in length and match one-for-one each amino acid as listed in order
from amino-terminus to carboxy-terminus. The tenth Shiga toxin
effector polypeptide in Table 6, 53-61-F2, comprises both a partial
replacement and an insertion of one amino acid at position 61 which
shifts the remaining carboxy-terminal, wild-type, amino acid
residues by one position. The eleventh Shiga toxin effector
polypeptide in Table 6 is a complete insertion of the entire T-cell
epitope between natively positioned amino acid residues 245 and
246. This insertion lies within B-cell epitope region #9 natively
positioned at amino acids 243-259 of SLT-1A.
[0460] Computational analysis in silico predicted that at least one
B-cell epitope present in the wild-type Shiga toxin was eliminated
for eight of the T-cell epitope embedded or inserted Shiga toxin
effector polypeptide variants, and no new B-cell epitopes were
predicted to be generated by embedding or inserting a T-cell
epitope in any of the exemplary Shiga toxin effector polypeptides
in Table 6 (see also Example 3, infra).
[0461] In addition, the Shiga toxin effector polypeptides
represented by SEQ ID NOs:11-17 and 19-21 each comprise a
disruption of a predicted endogenous CD4+ T-cell epitope(s).
Diphtheria Toxin Derived Effector Polypeptides
[0462] Similar to the above descriptions of modifying Shiga
toxin-derived polypeptides, T-cell epitopes were embedded into
diphtheria toxin-derived polypeptides with proteasome delivery
effector function to create exemplary T-cell epitope embedded,
diphtheria toxin effector polypeptides of the invention. The T-cell
epitopes were selected from a peptide in Table 1 and embedded to
disrupt at least one predicted B-cell epitope region described in
Table 5.
[0463] All the diphtheria toxin-derived polypeptides of this
example comprised the catalytic domain from the diphtheria toxin A
Subunit continuous with the translocation domain from the
diphtheria toxin B Subunit, a furin cleavage motif between the A
and B subunit derived, toxin effector polypeptide regions, and a
predicted disulfide bond between cysteines in the A and B subunit
derived, toxin effector polypeptide regions. Thus, the diphtheria
toxin-derived polypeptides in this example comprise both a
proteasome delivery effector region and ribotoxic toxin effector
polypeptide. The polypeptide sequences of exemplary, T-cell epitope
embedded, diphtheria toxin effector polypeptides of the invention
are provided as SEQ ID NOs: 46, 47, and 48.
[0464] Using standard techniques, a series of mutations were made
in the diphtheria toxin effector polypeptide in order to embed a
T-cell epitope in a position overlapping a predicted B-cell epitope
region (see Table 5). Table 7 shows examples of T-cell epitope
embedded, diphtheria toxin effector polypeptides by denoting the
position of embedded T-cell epitope based on the native diphtheria
toxin polypeptide sequence in SEQ ID NO:44, the T-cell epitope
name, the T-cell epitope peptide sequence, the predicted B-cell
epitope region disrupted, and the replaced amino acid sequence in
the native diphtheria toxin polypeptide sequence.
TABLE-US-00010 TABLE 7 Exemplary Diphtheria Toxin Effector
Polypeptides with T-Cell Epitopes Embedded into B-Cell Epitope
Regions B-Cell Epitope Position B-Cell Prediction (native T-Cell
T-Cell Epitope Epitope B-Cell Epitope original neo- positions)
Epitope Embedded Region Region Replaced epitope epitope 34-42 F2
GILGFVFTL 2 GIQKPKSGT eliminated none 69-77 C2 NLVPMVATV 3
NENPLSGKA eliminated none 168-176 F3 ILRGSVAHK 6 ETRGKRGQD
eliminated none
[0465] The T-cell epitope embedded, diphtheria toxin effector
polypeptide variants (SEQ ID NOs: 46, 47, and 48) were analyzed for
any change in the predicted B cell epitopes as described above. In
all three T-cell epitope embedded, diphtheria toxin effector
polypeptide variants, the predicted B-cell epitope in the wild-type
diphtheria toxin amino acid sequence was eliminated, and no new
B-cell epitopes were predicted (Table 7).
[0466] Three T-cell epitope embedded, diphtheria toxin effector
polypeptide variants (SEQ ID NOs: 46, 47, and 48), and the parental
diphtheria toxin effector polypeptide comprising only wild-type
toxin amino acid sequences (SEQ ID NO:45), were each designed with
an amino-terminal methionine and a carboxy-terminal
polyhistidine-tag (6.times.His tag) to facilitate expression and
purification. Both exemplary T-cell epitope embedded, diphtheria
toxin effector polypeptide variants of the invention and the
parental diphtheria toxin effector polypeptide comprising only
wild-type toxin amino acid sequences were produced by a bacterial
expression system known in the art and purified under conditions
known in the art, such as, e.g., nickel-nitrilotriacetic acid
(Ni-NTA) resin chromatography.
E. Generating Shiga Toxin Effector Polypeptides with Embedded
T-Cell Epitopes which do not Disrupt any B-Cell Epitope Region
[0467] Recognizing all the B-cell epitope region predictions from
all the methods described in the Examples (Table 2), regions of
SLT-1A that were not predicted to contain any B-cell epitope were
identified. T-cell epitope peptide sequences from Table 1 are
embedded in those regions identified to lack B-cell epitopes by
replacing the native amino acids by substitutions to create three
different exemplary Shiga toxin effector polypeptides of the
invention as shown in Table 8. Table 8 shows the identified regions
in the mature, native SLT-1A polypeptide sequence and the
replacement T-cell epitope sequences constructed into the Shiga
toxin effector polypeptides (see SEQ ID NOs: 22-39).
TABLE-US-00011 TABLE 8 T-Cell Epitopes Embedded Outside B-Cell
Epitope Regions in Shiga Toxin Effector Polypeptides Position
T-Cell T-Cell (native residue Epitope Epitope WT Region positions)
Name Embedded replaced 66-74 F2 GILGFVFTL NLRLIVERN 75-83 F2
GILGFVFTL NLYVTGFVN 157-165 F2 GILGFVFTL AMLRFVTVT 164-172 F2
GILGFVFTL VTAEALRFR 221-229 F2 GILGFVFTL VGRISFGSI 231-239 F2
GILGFVFTL AILGSVALI 66-74 F3 ILRGSVAHK NLRLIVERN 75-83 F3 ILRGSVAHK
NLYVTGFVN 157-165 F3 ILRGSVAHK AMLRFVTVT 164-172 F3 ILRGSVAHK
VTAEALRFR 221-229 F3 ILRGSVAHK VGRISFGSI 231-239 F3 ILRGSVAHK
AILGSVALI 66-74 C2 NLVPMVATV NLRLIVERN 75-83 C2 NLVPMVATV NLYVTGFVN
157-165 C2 NLVPMVATV AMLRFVTVT 164-172 C2 NLVPMVATV VTAEALRFR
221-229 C2 NLVPMVATV VGRISFGSI 231-239 C2 NLVPMVATV AILGSVALI
[0468] The Shiga toxin effector polypeptide sequences comprising,
as exact replacements, the embedded T-cell epitopes in Table 8 were
analyzed using the BcePred program. None of the embedded T-cell
epitope exact replacements in Table 8 disrupted any of the six
epitope regions predicted by that program. One of the embedded
T-cell epitope replacement sequences in Table 8, variant 75-83-F3,
resulted in the prediction of a new B-cell epitope. Embedding
T-cell epitopes near the regions (66-74) and/or (157-165) may
interfere with the Shiga toxin effector function of catalytic
activity because of their proximity to at least one amino acid
known to be required for SLT-1A catalytic activity (e.g. Y77 and
E167).
[0469] In addition, the Shiga toxin effector polypeptides
represented by SEQ ID NOs: 22-39 all comprise a disruption of a
predicted endogenous CD4+ T-cell epitope(s) except for the
polypeptides with heterologous T-cell epitopes embedded at position
221-229, which are represented by SEQ ID NOs: 26, 32, and 38.
F. Generating Toxin Effector Polypeptides with Embedded T-Cell
Epitopes which Disrupt Toxin Catalytic Function
[0470] The most critical residues for enzymatic activity of the
Shiga toxin A Subunits include tyrosine-77 (Y77) and glutamate-167
(E167) (Di, Toxicon 57: 535-39 (2011)). T-cell epitope peptide
sequences from Table 1 are embedded into Shiga toxin effector
polypeptides such that either Y77 or E167 is mutated in order to
reduce or eliminate Shiga toxin enzymatic activity. Six different
exemplary Shiga toxin effector polypeptides of the invention
comprising a heterologous T-cell epitope disrupting a catalytic
amino acid residue are shown in Table 9. Table 9 shows the position
of the embedded T-cell epitopes in the mature, native SLT-1A
polypeptide sequence, the replacement T-cell epitope sequences
which are embedded, the replaced sequences in the mature, native
SLT-1A polypeptide sequence, and a resulting catalytic residue
disruption (see also SEQ ID NOs: 23, 29, 40, 41, 42, and 43).
TABLE-US-00012 TABLE 9 T-Cell Epitopes Embedded in Shiga Toxin
Effector Polypeptides to Inactivate Shiga Toxin Catalytic Function
Position (native T-Cell T-Cell Catalytic residue Epitope Epitope WT
Region Residue positions) name Embedded Replaced Change 75-83 C2
NLVPMVATV NLYVTGFVN Y77V 75-83 F3 ILRGSVAHK NLYVTGFVN Y77R 77-85 F2
GILGFVFTL YVTGFVNRT Y77G 159-167 F2 GILGFVFTL LRFVTVTAE E167L
159-167 F3 ILRGSVAHK LRFVTVTAE E167K 162-170 C2 NLVPMVATV VTVTAEALR
E167V
[0471] All of the Shiga toxin effector polypeptides represented by
SEQ ID NOs: 23, 29, 40, 42, and 43 comprise disruptions of a
predicted endogenous CD4+ T-cell epitope(s). In addition, among the
exemplary Shiga toxin effector polypeptides with embedded T-cell
epitopes which do not disrupt any B-cell epitope region shown in
Table 8, at least eight of them disrupt a catalytic amino acid
residue of the Shiga toxin effector region (see SEQ ID NOs: 23, 25,
29, 31 35, and 37).
[0472] In addition to embedding and inserting at a single site,
multiple immunogenic T-cell epitopes for MHC class I presentation
are embedded and/or inserted within the same Shiga toxin-derived
polypeptides or diphtheria toxin-derived polypeptides for use in
the targeted delivery of a plurality of T-cell epitopes
simultaneously, such as, e.g., disrupting a B-cell epitope region
with a first embedded T-cell epitope and disrupting a toxin
catalytic function with a second embedded T-cell epitope. However,
it should be noted that a single embedded T-cell epitope can
simultaneously disrupt both a B-cell epitope region and a toxin
catalytic function.
Example 2
Testing Toxin-Derived Effector Polypeptides for Retention of
Ribotoxic Toxin Effector Function
[0473] Exemplary toxin-derived effector polypeptides of the
invention were tested for retention of ribotoxic toxin effector
function.
Shiga Toxin Derived Effector Polypeptides' Retention of
Ribotoxicity
[0474] The retention of the enzymatic activity of the parental
Shiga toxin effector polypeptide after embedding or inserting one
or more T-cell epitopes was determined using a ribosome inhibition
assay. The results of this assay in this example were based on
performing the assay with each Shiga toxin effector polypeptide as
a component of a cytotoxic protein. The specific cytotoxicities of
different cytotoxic proteins comprising different Shiga toxin
effector polypeptides were measured using a tissue culture
cell-based toxicity assay. The enzymatic and cytotoxic activities
of the exemplary cytotoxic, cell-targeted proteins of the invention
were compared to the parental Shiga toxin effector polypeptide
alone (no cell-targeting binding region) and a "WT" cytotoxic
protein comprising the same cell-targeting domain (e.g. binding
region comprising an immunoglobulin-type binding region capable of
binding an extracellular target biomolecule with high affinity) but
with a wild-type Shiga toxin effector region (WT).
[0475] The ribosome inactivation capabilities of cytotoxic proteins
comprising embedded or inserted T-cell epitopes were determined
using a cell-free, in vitro protein translation assay using the
TNT.RTM. Quick Coupled Transcription/Translation kit (L1170 Promega
Madison, Wis., U.S.). The kit includes Luciferase T7 Control DNA
(L4821 Promega Madison, Wis., U.S.) and TNT.RTM. Quick Master Mix.
The ribosome activity reaction was prepared according to
manufacturer's instructions. A series of 10-fold dilutions of the
protein to be tested, comprising either a mutated Shiga toxin
effector polypeptide region or WT region, was prepared in an
appropriate buffer and a series of identical TNT reaction mixture
components were created for each dilution. Each sample in the
dilution series was combined with each of the TNT reaction mixtures
along with the Luciferase T7 Control DNA. The test samples were
incubated for 1.5 hours at 30 degrees Celsius (.degree. C.). After
the incubation, Luciferase Assay Reagent (E1483 Promega, Madison,
Wis., U.S.) was added to all test samples and the amount of
luciferase protein translation was measured by luminescence
according to manufacturer's instructions. The level of
translational inhibition was determined by non-linear regression
analysis of log-transformed concentrations of total protein versus
relative luminescence units. Using statistical software (GraphPad
Prism, San Diego, Calif., U.S.), the half maximal inhibitory
concentration (IC.sub.50) value was calculated for each sample
using the Prism software function of log(inhibitor) vs. response
(three parameters) [Y=Bottom+((Top-Bottom)/(1+10 (X-Log
IC.sub.50)))] under the heading dose-response-inhibition. The
IC.sub.50 values were calculated for each de-immunized protein
comprising a B cell epitope replacement/disruption Shiga toxin
effector polypeptide region and a control protein comprising a
wild-type Shiga toxin effector region (WT).
[0476] The exemplary Shiga toxin effector polypeptide regions of
the invention exhibited ribosome inhibition comparable to a
wild-type Shiga toxin effector polypeptide (WT) as indicated in
Table 10. As reported in Table 10, any construct comprising a Shiga
toxin effector polypeptide of the invention which exhibited an
IC.sub.50 within 10-fold of the positive control construct
comprising a wild-type Shiga toxin effector region was considered
to exhibit ribosome inhibition activity comparable to
wild-type.
TABLE-US-00013 TABLE 10 Retention of Shiga Toxin Function(s): In
vitro catalytic activity and in vivo specific cytotoxicity of
exemplary Shiga toxin effector polypeptides Exemplary Shiga Toxin
Shiga Toxin Functions Effector Polypeptide Ribosome
Position-T-Cell-Epitope Inactivation Specific Cytotoxicity
4-12-F3-4 comparable to WT comparable to WT 43-51-C2 comparable to
WT comparable to WT 44-52-F2 comparable to WT comparable to WT
53-61-F2 comparable to WT comparable to WT 53-61-F2-2 comparable to
WT comparable to WT 53-61-F2-3 comparable to WT comparable to WT
53-61-C2 comparable to WT comparable to WT 104-112-C2 comparable to
WT comparable to WT 180-188-F2-4 comparable to WT comparable to WT
245-F3 comparable to WT comparable to WT
[0477] The retention of cytotoxicity by exemplary Shiga toxin
effector polypeptides of the invention after T-cell epitope
embedding/insertion was determined by a cell-kill assay in the
context of the Shiga toxin effector polypeptide as a component of a
cytotoxic protein. The cytotoxicity levels of proteins comprising
Shiga toxin effector polypeptides, comprising an embedded or
inserted T-cell epitope, were determined using extracellular target
expressing cells as compared to cells that do not express a target
biomolecule of the cytotoxic protein's binding region. Cells were
plated (2.times.10.sup.3 cells per well for adherent cells, plated
the day prior to protein addition or 7.5.times.10.sup.3 cells per
well for suspension cells, plated the same day as protein addition)
in 20 .mu.L cell culture medium in 384-well plates. A series of
10-fold dilutions of each protein comprising a mutated Shiga toxin
effector polypeptide region to be tested was prepared in an
appropriate buffer, and then 5 .mu.L of the dilutions or buffer
control were added to the cells. Control wells containing only
media were used for baseline correction. The cell samples were
incubated with the proteins or just buffer for 3 days at 37.degree.
C. and in an atmosphere of 5% carbon dioxide (CO.sub.2). The total
cell survival or percent viability was determined using a
luminescent readout using the CellTiter-Glo.RTM. Luminescent Cell
Viability Assay (G7573 Promega Madison, Wis., U.S.) according to
the manufacturer's instructions.
[0478] The Percent Viability of experimental wells was calculated
using the following equation: (Test RLU-Average Media RLU)/(Average
Cells RLU-Average Media RLU)*100. Log polypeptide concentration
versus Percent Viability was plotted in Prism (GraphPad Prism, San
Diego, Calif., U.S.) and log (inhibitor) versus response (3
parameter) analysis was used to determine the half-maximal
cytotoxic concentration (CD.sub.50) value for the tested proteins.
The CD.sub.50 was calculated for each protein comprising an
exemplary Shiga toxin effector polypeptide of the invention in
Table 10, positive-control cytotoxic protein comprising a wild-type
Shiga toxin effector region, and the wild-type SLT-1 A Subunit
alone (no targeting domain)--both were considered WT positive
controls.
[0479] The protein comprising exemplary Shiga toxin effector
polypeptides of the invention exhibited cell-specific
cytotoxicities comparable to a wild-type (WT) Shiga toxin effector
polypeptide as indicated in Table 10. As reported in Table 10 with
regard to specific cytotoxicity, "comparable to WT" means a protein
comprising a Shiga toxin effector polypeptide, comprising an
embedded or inserted T-cell epitope, exhibited a CD.sub.50 to a
target positive cell population within 10-fold of a protein
comprising a wild-type (WT) Shiga toxin effector region and/or less
than 50-fold of the SLT-1A subunit alone.
[0480] In addition, the same protein constructs comprising
exemplary Shiga toxin effector polypeptides of the invention
exhibited specific cytotoxicity to biomolecular-target-expressing
cells as compared to biomolecular-target-negative cells (i.e. cells
which did not express, at a cellular surface, the biomolecular
target of the cell-target binding region of the protein construct).
Thus, all the proteins comprising the exemplary Shiga toxin
effector polypeptides in Table 10 were cytotoxic proteins
exhibiting Shiga toxin effector functions comparable to wild-type
(WT), and each cytotoxic protein comprised a disruption in one or
more predicted, B-cell epitope regions.
Diphtheria Toxin Derived Effector Polypeptides' Retention of
Ribotoxicity
[0481] The catalytic activity of exemplary, T-cell epitope
embedded, diphtheria toxin effector polypeptides were compared to
diphtheria toxin effector polypeptides comprising only wild-type
amino acid sequences, referred to herein as "wild-type" or "WT."
Both T-cell epitope embedded, diphtheria toxin effector polypeptide
variants retained ribosome inactivation activity.
[0482] The retention of enzymatic activity of diphtheria toxin
effector polypeptide variants with embedded T-cell epitopes in the
context of a cell-targeted molecule was tested using a ribosome
inhibition assay and a wild-type (WT) diphtheria toxin effector
polypeptide as a positive control. The ribosome inactivation
capabilities of these toxin effector polypeptides was determined
using a cell-free, in vitro protein translation assay using the
TNT.RTM. Quick Coupled Transcription/Translation kit (L1170 Promega
Madison, Wis., U.S.) as described above unless otherwise noted.
First, the diphtheria toxin effector polypeptides were cleaved in
vitro with furin (New England Biolabs, Ipswich, Mass., U.S.) under
standard conditions. Then the cleaved proteins were diluted in
buffer to make a series of dilutions for each sample. Each dilution
in each series was combined with each of the TNT reaction mixtures
along with the Luciferase T7 Control DNA and tested for ribosome
inactivation activity as described above.
[0483] The IC.sub.50 was calculated, as described above, for the
diphtheria toxin effector polypeptides. FIG. 2 and Table 11 show
the results of this in vitro assay for retention of diphtheria
ribotoxic toxin effector function by exemplary, T-cell embedded,
diphtheria toxin effector polypeptides of the invention. The
activity of the T-cell embedded, diphtheria toxin effector
polypeptides was comparable to the wild-type (WT) positive control
because the IC.sub.50 values were within ten-fold of the wild-type
diphtheria toxin effector polypeptide control (FIG. 2; Table
11).
TABLE-US-00014 TABLE 11 Retention of catalytic activity by
exemplary T-cell epitope embedded, Diphtheria toxin effector
polypeptides Fold Change Diphtheria Toxin Effector Polypeptide
IC.sub.50 (.mu.M) from WT Wild-type (WT) 1.80 1.0 T-cell epitope
embedded, diphtheria toxin 4.94 2.8 effector polypeptide variant
34-42-F2 T-cell epitope embedded, diphtheria toxin 13.3 7.5
effector polypeptide variant 168-176-F3 Exemplary Diphtheria Toxin
Effector Diphtheria Toxin Function: Polypeptide Ribosome
Inactivation 34-42-F2 comparable to WT 168-176-F3 comparable to
WT
Example 3
Testing the De-Immunization Effects of Disruption of B-Cell Epitope
Regions and CD4+ T-Cell Epitope Regions in T-Cell Epitope Embedded,
Toxin Effector Polypeptides
[0484] The disruption of B-cell epitope regions in Shiga toxin
effector polypeptides using embedded or inserted T-cell epitopes
was tested for de-immunization by investigating levels of
antigenicity and/or immunogenicity compared to wild-type (WT) Shiga
toxin effector polypeptides comprising only wild-type amino acid
sequences.
Testing De-Immunization Via Western Analysis
[0485] To analyze de-immunization, the antigenicity or
immunogenicity levels of Shiga toxin effector polypeptides was
tested both in silico and by Western blotting using pre-formed
antibodies which recognize wild-type Shiga toxin effector
polypeptides.
[0486] Each Shiga toxin effector polypeptide described in Table 6
(SEQ ID NOs: 11-21) was checked for the disruption of predicted
B-cell epitopes using the BcePred webserver using the following
parameters: flexibility readout with the default settings of
hydrophilicity 2, accessibility 2, exposed surface 2.4, antigenic
propensity 1.8, flexibility 1.9, turns 1.9, polarity 2.3, and
combined 1.9 (Saha S, Raghava G, Lecture Notes in Comput Sci 3239:
197-204 (2004)). Three predicted immunogenic epitope regions
identified in the wild-type SLT-1 A Subunit by other programs (see
Table 2) were not predicted by the BcePred flexibility approach
with the default settings and, thus, could not be analyzed.
[0487] The T-cell epitope embedding or insertion in the following
exemplary Shiga toxin effector polypeptides of the invention SEQ ID
NOs: 11-21 resulted in the elimination of the predicted B-cell
epitope intended for disruption without the introduction of any
epitopes de novo (neo-epitopes) (Table 12). None of the tested
exemplary Shiga toxin effector polypeptides of the invention
resulted in the generation of any de novo predicted B-cell epitopes
using the BcePred flexibility approach with the default settings
(Table 12). Any B-cell epitope region not predicted by the BcePred
flexibility approach with the default settings was indicated with
"not identified" and the result after T-cell epitope embedding or
insertion was indicated with "N/A" to denote "not applicable.
TABLE-US-00015 TABLE 12 Analysis of B-Cell Epitope Region
Disruption by Embedded or Inserted T-Cell Epitopes B-Cell Epitope
Region Disruption BcePred Flexibility B-Cell with T-Cell Epitope
Replacement Epitope Predictions T-Cell T-Cell B-Cell WT Shiga
Modified Epitope Epitope Epitope Region Toxin Sequence Shiga Toxin
Neo-Epitope Position Embedded Disrupted (parental) Sequence
Prediction 4-12 ILRFSVAHK 1 not N/A none identified 43-51 NLVPMVATV
3 39-46 eliminated none 44-52 GILGFVFTL 3 39-46 eliminated none
44-52 NLVPMVATV 3 39-46 eliminated none 53-61 GILGFVFTL 4 55-61
eliminated none 53-61 DILGFVFTL 4 55-61 eliminated none 53-61
DILGFDFTL 4 55-61 eliminated none 53-61 NLVPMVATV 4 55-61
eliminated none 104-112 NLVPMVATV 5 105-111 eliminated none 180-188
GILGDVFTL 7 181-187 eliminated none 245/246 ILRFSVAHK 9 not N/A
none identified
[0488] The relative antigenicity levels of Shiga toxin effector
polypeptides was tested for de-immunization by Western blotting
using pre-formed antibodies, both polyclonal and monoclonal
antibodies, which recognize the wild-type (WT) Shiga toxin effector
polypeptides comprising amino acids 1-251 of SEQ ID NO:1.
[0489] Western blotting was performed on cytotoxic proteins
comprising a Shiga toxin effector polypeptide comprising either
only a wild-type (WT) Shiga toxin sequence or one of various
modified Shiga toxin sequences comprising a B-cell epitope region
disruption via replacement with a T-cell epitope (SEQ ID NO:
11-19). These cytotoxic proteins were loaded in equal amounts to
replicate, 4-20% sodium dodecyl sulfate (SDS), polyacrylamide gels
(Lonza, Basel, CH) and electrophoresed under denaturing conditions.
The resulting gels were either analyzed by Coomassie staining or
transferred to polyvinyl difluoride (PVDF) membranes using the
iBlot.RTM. (Life Technologies, Carlsbad, Calif., U.S.) system
according to manufacturer's instructions. The resulting membranes
were probed under standard conditions using the following
antibodies: rabbit polyclonal .alpha.-NWSHPQFEK (A00626, Genscript,
Piscataway, N.J., U.S.) which recognizes the polypeptide NWSHPQFEK
also known as Streptag.RTM. II, mouse monoclonal .alpha.-Stx (mAb1
or anti-SLT-1A mAb1) (BEI NR-867 BEI Resources, Manassas, Va.,
U.S.; cross reactive with Shiga toxin and Shiga-like toxin 1 A
subunits), rabbit polyclonal antibody .alpha.-SLT-1A (pAb1 or
anti-SLT-1A pAb1) (Harlan Laboratories, Inc. Indianapolis, Ind.,
U.S., custom antibody production raised against the SLT-1A amino
acids 1-251), and rabbit polyclonal antibody .alpha.-SLT-1A (pAb2
or anti-SLT-1A pAb2) (Genscript, Piscataway, N.J., U.S., custom
antibody production), which was raised against the peptides
RGIDPEEGRFNN and HGQDSVRVGR. The peptide sequence RGIDPEEGRFNN is
located at amino acids 55-66 in SLT-1A and StxA, spanning a
predicted B cell epitope, and the peptide sequence HGQDSVRVGR is
located at 214-223 in SLT-1A and StxA, spanning a predicted B-cell
epitope.
[0490] Membrane bound antibodies were detected using standard
conditions and, when appropriate, using horseradish peroxidase
(HRP) conjugated secondary antibodies (goat anti-rabbit-HRP or goat
anti-mouse-HRP, Thermo Scientific, Rockford, Ill., U.S.). FIGS. 3-4
show images of Western blots with the lanes of the gels and/or
membranes numbered and the figure legends indicate by the same
respective numbering which Shiga toxin effector polypeptide was a
component of the cytotoxic protein sample loaded into each lane.
For each gel, the Coomassie staining and/or anti-streptag II
Western blot signal serve as total cytotoxic protein loading
controls. All the modified Shiga toxin effector polypeptides had
reduced or abolished recognition by one or more antibodies that can
recognize wild-type SLT-1A indicating a reduced antigenicity and
successful de-immunization. The result of the Western blot analyses
shown in FIGS. 3 and 4 are summarized in Table 13.
TABLE-US-00016 TABLE 13 Epitope Disruption Analysis by Western:
Exemplary Shiga toxin effector polypeptides tested show reduced or
abolished antibody binding Western Blot Result anti-SLT- anti-SLT-
anti-SLT- Cytotoxic Protein comprising: 1A pAb1 1A pAb2 1A mAb1 WT
Shiga toxin effector region present present present Exemplary Shiga
toxin effector polypeptide comprising a B-Cell epitope region
disrupted with the T-cell epitope below: B-Cell T-Cell T-Cell
Epitope Epitope Epitope Region Position Embedded Disrupted 4-12
ILRFSVAHK 1 reduced present abolished 43-51 NLVPMVATV 3 reduced
present abolished 44-52 GILGFVFTL 3 strongly reduced abolished
reduced 53-61 GILGFVFTL 4 reduced abolished abolished 53-61
DILGFVFTL 4 reduced abolished not tested 53-61 DILGFDFTL 4 reduced
abolished not tested 53-61 NLVPMVATV 4 strongly strongly abolished
reduced reduced 104-112 NLVPMVATV 5 present present abolished
180-188 GILDDVFTL 7 present present abolished
Testing CD4+ T-Cell De-Immunization
[0491] Disruptions in predicted CD4+ T-cell epitope regions are
tested for reductions in CD4+ T-cell immunogenicity using assays of
human CD4+ T-cell proliferation in the presence of exogenously
administered polypeptides and assays of human CD4+ dendritic T-cell
stimulation in the presence of human monocytes treated with
administered polypeptides.
[0492] T-cell proliferation assays known to the skilled worker are
used to test the effectiveness of CD4+ T-cell epitope
de-immunization in exemplary toxin effector polypeptides comprising
T-cell epitopes embedded or inserted into predicted CD4+ T-cell
epitopes. The T-cell proliferation assay of this example involves
the labeling of CD4+ T-cells and then measuring changes in
proliferation using flow cytometric methods in response to the
administration of different peptides derived from either a
polypeptide de-immunized using the methods of embedding or
inserting a heterologous CD8+ T-cell epitope (e.g., SEQ ID NOs:
11-43) or a reference polypeptide that does not have any
heterologous T-cell epitope associated with it.
[0493] A series of overlapping peptides derived from a polypeptide
are synthesized and tested in the CFSE CD4+ T cell proliferation
assay (ProImmune Inc., Sarasota, Fla., U.S). Human CD8+ T-cell
depleted, peripheral blood mononuclear cells (PBMCs) labeled with
CFSE are cultured with 5 .mu.M of each peptide of interest for
seven days in six replicate wells. Each assay plate includes a set
of untreated control wells. The assay also incorporates reference
antigen controls, comprising synthetic peptides for known MHC class
II antigens.
[0494] The CD8+ T-cell depleted, PBMCs that proliferate in response
to an administered peptide will show a reduction in CFSE
fluorescence intensity as measured directly by flow cytometry. For
a naive T-cell analysis, the Percentage Stimulation above
background is determined for each stimulated sample, through
comparison with results from an unstimulated sample, such as by
ranking with regard to fluorescent signal, as negative, dim, or
high. Counts for the CD4+CFSE T-cell dim population in each sample
are expressed as a proportion of the total CD4+ T-cell population.
The replicate values are used to calculate Percentage Stimulation
above Background (proportion of CD4+ T-cell CFSE dim cells with
antigen stimulation, minus proportion of CD4+ T-cell CFSE dim cells
without antigen stimulation). The mean and standard error of the
mean are calculated from the replicate values. A result is
considered "positive" if the Percentage Stimulation above
background is greater than 0.5% and also greater than twice the
standard error above background. To allow for comparison of
peptides, a Response Index is calculated. This index is based on
multiplying the magnitude of response (Percentage Stimulation above
background) for each peptide by the number of responding donors
(Percentage Antigenicity) for each peptide.
Determining Relative CD4+ T-Cell Immunogenicity
[0495] The relative CD4+ T-cell immunogenicity of exemplary,
full-length polypeptides of the invention is determined using the
following dendritic cell (DC) T-cell proliferation assay. This DC
T-cell assay measures CD4+ T-cell responses to exogenously
administered polypeptides or proteins. The DC T-cell assay is
performed using ProImmune's DC-T assay service to determine the
relative levels of CD4+ T-cell driven immunogenicity between
polypeptides, proteins, and cell-targeted molecules of the
invention as compared to the starting parental polypeptides,
proteins, or cell-targeted molecules which lack the addition of any
heterologous T-cell epitope. The DC T-cell assay of this example
involves testing human dendritic cells for antigen presentation of
peptides derived from the administered polypeptide, protein, or
cell-targeted molecule samples.
[0496] Briefly, healthy human donor tissues are used to isolate
typed samples based on high-resolution MHC Class II tissue-typing.
A cohort of 20, 40 or 50 donors is used. First, monocytes obtained
from human donor PBMCs are cultured in a defined medium to generate
immature dendritic cells. Then, the immature dendritic cells are
stimulated with a well-defined control antigen and induced into a
more mature phenotype by further culture in a defined medium. Next,
CD8+ T-cell depleted donor PBMCs from the same human donor sample
are labeled with CFSE. The CFSE-labeled, CD8+ T-cell depleted PBMCs
are then cultured with the antigen-primed, dendritic cells for
seven days to allow for CD4+ dendritic cell stimulation, after
which eight replicates for each sample are tested. As negative
controls, each dendritic cell culture series also includes a set of
untreated dendritic cells. For a positive control, the assay
incorporates two well-defined reference antigens, each comprising a
full-length protein.
[0497] To evaluate dendritic cell based immunogenicity, the
frequency of donor cell responses is analyzed across the study
cohort. Positive responses in the assay are considered indicative
of a potential in vivo CD4+ T-cell response. A positive response,
measured as a percentage of stimulation above background, is
defined as percentages greater than 0.5 percent (%) in 2 or more
independent donor samples. The strength of positive donor cell
responses is determined by taking the mean percentage stimulation
above background obtained across accepted donors for each sample. A
Response Index is calculated by multiplying the value of the
strength of response by the frequency of the donors responding to
determine levels of CD4+ T-cell immunogenicity for each sample. In
addition, a Response index, representing the relative CD4+ T-cell
immunogenicity is determined by comparing the results from two
samples, one comprising a CD8+ T cell epitope embedded in a
predicted CD4+ T-cell epitope region and a second variant which
lacks any disruption to the same predicted CD4+ T-cell region to
determine if the disruption reduces the CD4+ T-cell response of
human donor cells.
Testing De-Immunization Via Relative Immunogenicity In Vivo
[0498] The relative immunogenicity levels of Shiga toxin effector
polypeptides are tested for de-immunization using mammalian models
of the human immune system. Mice are intravenously administered
cytotoxic proteins or polypeptides comprising either wild-type (WT)
or de-immunized forms of the Shiga toxin effector polypeptide
component 3 times per week for two weeks or more. Blood samples are
taken from the injected mice and tested by enzyme-linked
immunosorbent assay (ELISA) for reactivity to the cytotoxic
proteins and/or the Shiga toxin effector polypeptide. Reduced
immunogenic responses will be elicited in mice injected with the
de-immunized Shiga toxin effector polypeptide, or compositions
comprising the same, as compared to mice injected only with the
wild-type form of the Shiga toxin effector polypeptide, or
composition comprising the same. The relatively reduced immunogenic
response will indicate that the de-immunized Shiga toxin effector
polypeptides are de-immunized with regard to having reduced
immunogenic potential after administration to a mammal and allowing
time for the mammal's immune system to respond.
[0499] In addition, diphtheria toxin effector polypeptides of the
invention (e.g. SEQ ID NOs: 46-48) are tested for de-immunization
using the methods of this example to verify the disruption of one
or more B-cell epitope regions in each diphtheria toxin effector
polypeptides comprising an embedded or inserted T-cell epitope.
Example 4
Testing Cellular Internalization, Sub-Cellular Routing, and
Presentation of an Embedded T-Cell Epitope on the Surfaces of
Target Cells by Exemplary Shiga Toxin Effector Polypeptides of the
Invention
[0500] In this example, the ability of exemplary cell-targeted
proteins of the invention, which each comprise an exemplary Shiga
toxin effector polypeptide of the invention, to deliver T-cell
epitopes to the MHC class I pathway of target cells for
presentation to the target cell surface was investigated. In
addition, cell-targeted proteins comprising diphtheria toxin
effector polypeptides of the invention (e.g. SEQ ID NOs: 46-48) are
tested using the methods of this example to verify their ability to
deliver embedded T-cell epitopes to the MHC class I presentation
system.
[0501] Using standard techniques known in the art, various
exemplary cell-targeted proteins of the invention were made where
each comprises a cell-type-targeting region and a Shiga toxin
effector polypeptide of the invention (see e.g. WO2014164680 and
WO2014164693). A cell-targeted protein of the invention comprises
both a Shiga toxin effector polypeptide of the invention and a
cell-targeting binding region capable of exhibiting high-affinity
binding to an extracellular target biomolecule physically-coupled
to the surface of a specific cell type(s). The cell-targeted
proteins of the invention are capable of selectively targeting
cells expressing the target biomolecule of their cell-targeting
binding region and internalizing into these target cells.
[0502] A flow cytometry method was used to demonstrate delivery and
extracellular display of a T-cell epitope (inserted or embedded in
a Shiga toxin effector region) in complex with MHC Class I
molecules on the surfaces of target cells. This flow cytometry
method utilizes soluble human T-cell receptor (TCR) multimer
reagents (Soluble T-Cell Antigen Receptor STAR.TM. Multimer, Altor
Bioscience Corp., Miramar, Fla., U.S.), each with high-affinity
binding to a different epitope-human HLA complex.
[0503] Each STAR.TM. TCR multimer reagent is derived from a
specific T-cell receptor and allows detection of a specific
peptide-MHC complex based on the ability of the chosen TCR to
recognize a specific peptide presented in the context of a
particular MHC class I molecule. These TCR multimers are composed
of recombinant human TCRs which have been biotinylated and
multimerized with streptavidin. The TCR multimers are labeled with
phycoerythrin (PE). These TCR multimer reagents allow the detection
of specific peptide-MHC Class I complexes presented on the surfaces
of human cells because each soluble TCR multimer type recognizes
and stably binds to a specific peptide-MHC complex under varied
conditions (Zhu X et al., J Immunol 176: 3223-32 (2006)). These TCR
multimer reagents allow the identification and quantitation by flow
cytometry of peptide-MHC class I complexes present on the surfaces
of cells.
[0504] The TCR CMV-pp65-PE STAR.TM. multimer reagent (Altor
Bioscience Corp., Miramar, Fla., U.S.) was used in this Example.
MHC class I pathway presentation of the CMV C2 peptide (NLVPMVATV)
by human cells expressing the HLA-A2 can be detected with the TCR
CMV-pp65-PE STAR.TM. multimer reagent which exhibits high affinity
recognition of the CMV-pp65 epitope-peptide (residues 495-503,
NLVPMVATV) complexed to human HLA-A2 and which is labeled with
PE.
[0505] The target cells used in this Example were immortalized
human cancer cells available from the ATCC (Manassas Va., U.S.).
Using standard flow cytometry methods known in the art, the target
cells were confirmed to express on their cell surfaces both the
HLA-A2 MHC-Class I molecule and the extracellular target
biomolecule of the cell-targeting moiety of the proteins used in
this Example.
[0506] The target cells were treated with the exemplary
cell-targeted proteins of the invention, each comprising different
Shiga toxin effector polypeptides comprising a T-cell epitope
embedded into a predicted B-cell epitope region. One of each of the
exemplary cell-targeted proteins of the invention tested in this
Example comprised one of the following Shiga toxin effector
polypeptides: 43-51-C2 (SEQ ID NO: 13), 53-61-C2(SEQ ID NO: 17),
and 104-112-C2(SEQ ID NO: 18). Sets of target cells were treated by
exogenous administration of the different exemplary cell-targeted
proteins of the invention at concentrations similar to those used
by others taking into account cell-type specific sensitivities to
Shiga toxins (see e.g. Noakes K et al., FEBS Lett 453: 95-9
(1999)). The treated cells were then incubated for six hours in
standard conditions, including at 37.degree. C. and an atmosphere
with 5% carbon dioxide, to allow for intoxication mediated by a
Shiga toxin effector region. Then the cells were washed with cell
culture medium, re-suspended in fresh cell culture medium, and
incubated for 20 hours prior to staining with the TCR CMV-pp65-PE
STAR.TM. multimer reagent.
[0507] As controls, sets of target cells were treated in three
conditions: 1) without any treatment ("untreated") meaning that no
exogenous molecules were added, 2) with exogenously administered
CMV C2 peptide (CMV-pp65, aa495-503: sequence NLVPMVATV,
synthesized by BioSynthesis, Lewisville, Tex., U.S.), and 3) with
exogenously administered CMV C2 peptide (NLVPMVATV, as above)
combined with a Peptide Loading Enhancer ("PLE," Altor Biosicence
Corp., Miramar, Fla.). The C2 peptide combined with PLE treatment
allowed for exogenous peptide loading and served as a positive
control. Cells displaying the appropriate MHC class I haplotype can
be forced to load the appropriate exogenously applied peptide from
an extracellular space (i.e. in the absence of cellular
internalization of the applied peptide) or in the presence of PLE,
which is a mixture of B2-microglobulin and other components.
[0508] After the treatments, all the sets of cells were washed and
incubated with the TCR CMV-pp65-PE STAR multimer reagent for one
hour on ice. The cells were washed and the fluorescence of the
samples was measured by flow cytometry using an Accuri.TM. C6 flow
cytometer (BD Biosciences, San Jose, Calif., U.S.) to detect the
presence of and quantify any TCR CMV-pp65-PE STAR.TM. multimer
bound to cells in the population (sometimes referred to herein as
"staining").
[0509] The results of the flow cytometric analysis of the sets of
differently treated cells are shown in FIG. 5 and Table 14. The
untreated control was used to identify the positive and negative
cell populations by employing a gate which results in less than 1%
of cells from the untreated control in the "positive" gate
(representing background signal). The same gate was then applied to
the other samples to characterize the positive population for each
sample. In FIG. 5, the flow cytometry histograms are given with the
counts (number of cells) on the Y-axis and the relative fluorescent
units (RFU) on the X-axis (log scale). The grey line in all
histograms shows the profile of the untreated cells and the black
line shows the profile of treated cells according to the treatment
indicated. In Table 14, the percentage of cells in a treatment set
which stained positive for the C2-epitope-peptide-HLA-A2 complex is
given. Positive cells in this assay were cells which were bound by
the TCR-CMV-pp65-PE STAR reagent and counted in the positive gate
described above. Table 14 also shows for each set the corresponding
indexed, mean, fluorescent intensity ("iMFI," the fluorescence of
the positive population multiplied by the percent positive) in
RFU.
TABLE-US-00017 TABLE 14 Flow Cytometry Results for Exemplary
Cell-targeted proteins of the invention: Peptide-epitope C2-MHC
class I complexes detected on the surfaces of intoxicated, target
cells TCR CMV-pp65-PE Flow Cytometry Target cell treatment:
exogenously Percentage of administered molecule(s) Positive Cells
iMFI (RFU) Untreated 0.96% 20 Cell-targeted protein with Shiga
toxin 7.6% 113 effector region 43-51-C2 Cell-targeted protein with
Shiga toxin 4.5% 64 effector region 53-61-C2 Cell-targeted protein
with Shiga toxin 6.7% 89 effector region 104-112-C2 C2 peptide only
0.95% 19 C2 peptide and PLE 36.7% 728
[0510] Cells administered with exogenous protein comprising
43-51-C2, 53-61-C2, and 104-112-C2 showed a positive signal for
cell-surface, C2-peptide-HLA-A2 complexes on 7.6%, 4.5%, and 6.7%
of the cells in their population, respectively. In contrast, cell
populations that were "untreated" and treated with "C2 peptide
only" contained less than 1% positive cells (0.96 and 0.95 percent,
respectively). Due to processing efficiency and kinetics, which
were not measured, it is possible that the percent of presented
C2-peptide-HLA-A2 complex detected at a single timepoint in a
"cell-targeted protein" treatment sample may not accurately reflect
the maximum presentation possible by these exemplary cell-targeted
proteins of the invention.
[0511] The positive control "C2 peptide and PLE" population
contained 36.7% positive cells; however, the peptide can only be
loaded onto the surface from an extracellular space ("exogenously")
and in the presence of PLE as shown by comparing with the "C2
peptide only" results which had a similar background staining level
(0.95%) as the untreated control.
[0512] The detection of the exogenously administered, embedded
T-cell epitope C2 complexed with human MHC Class I molecules (C2
epitope-peptide/HLA-A2) on the cell surface of intoxicated target
cells demonstrated that cell-targeted proteins comprising the
exemplary Shiga toxin effector regions 43-51-C2, 53-61-C2, or
104-112-C2 were capable of entering target cells, performing
sufficient sub-cellular routing, and delivering enough embedded
T-cell epitope to the MHC class I pathway for surface presentation
on the target cell surface.
Example 5
Testing Cytotoxic T-Cell Mediated Cytolysis of Intoxicated Target
Cells and Other Immune Responses Triggered by MHC Class I
Presentation of T-Cell Epitopes Delivered by Proteins of the
Present Invention
[0513] In this example, standard assays known in the art are used
to investigate the functional consequences of target cells' MHC
class I presentation of T-cell epitopes delivered by exemplary
cell-targeted proteins of the invention. The functional
consequences to investigate include CTL activation, CTL mediated
target cell killing, and CTL cytokine release by CTLs.
[0514] A CTL-based cytotoxicity assay is used to assess the
consequences of epitope presentation. The assay involves
tissue-cultured target cells and T-cells. Target cells are
intoxicated as described in Example 4. Briefly, target cells are
incubated for six hours in standard conditions with different
exogenously administered proteins, where certain proteins comprise
a Shiga toxin effector polypeptide of the invention or a diphtheria
toxin effector polypeptide of the invention. Next, CTLs are added
to the intoxicated target cells and incubated to allow for the
T-cells to recognize and bind any target-cells displaying
epitope-peptide/MHC class I complexes. Then certain functional
consequences are investigated using standard methods known to the
skilled worker, including CTL binding to target cells, target cell
killing by CTL-mediated cytolysis, and the release of cytokines,
such as interferon gamma or interleukins by ELISA or ELIspot.
[0515] The activation of CTLs by target cells displaying
epitope-peptide/MHC class I complexes is quantified using
commercially available CTL response assays, e.g. CytoTox96.RTM.
non-radioactive assays (Promega, Madison, Wis., U.S.), Granzyme B
ELISpot assays (Mabtech, Inc., Cincinnati, Ohio, U.S.), caspase
activity assays, and LAMP-1 translocation flow cytometric assays.
To specifically monitor CTL-mediated killing of target cells,
carboxyfluorescein succinimidyl ester (CFSE) is used to
target-cells for in vitro and in vivo investigation as described in
the art (see e.g. Durward M et al., J Vis Exp 45 pii 2250
(2010)).
Example 6
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Specific to CD20 (.alpha.CD20 Fused with
SLT-1A)
[0516] In this example, a T-cell hyper-immunized and B-cell/CD4+
T-cell de-immunized Shiga toxin effector region is derived from the
A subunit of Shiga-like Toxin 1 (SLT-1A) as described above. An
immunoglobulin-type binding region .alpha.CD20-antigen is derived
from an immunoglobulin-type domain recognizing human CD20 (see e.g.
Haisma et al., Blood 92: 184-90 (1999); Geng S et al., Cell Mol
Immunol 3: 439-43 (2006); Olafesn T et al., Protein Eng Des Sel 23:
243-9 (2010)), which comprises an immunoglobulin-type binding
region capable of binding an extracellular part of CD20. CD20 is
expressed on multiple cancer cell types, such as, e.g., B-cell
lymphoma cells, hairy cell leukemia cells, B-cell chronic
lymphocytic leukemia cells, and melanoma cells. In addition, CD20
is an attractive target for therapeutics to treat certain
autoimmune diseases, disorders, and conditions involving overactive
B-cells.
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.CD20
[0517] The immunoglobulin-type binding region .alpha.CD20 and Shiga
toxin effector region (such as, e.g., SEQ ID NOs: 11-43) are linked
together. For example, a fusion protein is produced by expressing a
polynucleotide encoding the .alpha.CD20-antigen-binding protein
SLT-1A::.alpha.CD20 (see, e.g., SEQ ID NOs: 49, 50, and 51).
Expression of the SLT-1A::.alpha.CD20 cytotoxic protein is
accomplished using either bacterial and/or cell-free, protein
translation systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.CD20
[0518] The binding characteristics, the maximum specific binding
(B.sub.max) and equilibrium binding constants (K.sub.D), of the
cytotoxic protein of this example for CD20+ cells and CD20- cells
is determined by fluorescence-based, flow-cytometry. The B.sub.max
for SLT-1A::.alpha.CD20 to CD20+ cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to CD20- cells
in this assay.
[0519] The ribosome inactivation abilities of the
SLT-1A::.alpha.CD20 cytotoxic protein is determined in a cell-free,
in vitro protein translation as described above in the previous
examples. The inhibitory effect of the cytotoxic protein of this
example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.CD20 on protein synthesis in this
cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.CD20 Using a CD20+ Cell-Kill Assay
[0520] The cytotoxicity characteristics of SLT-1A::.alpha.CD20 are
determined by the general cell-kill assay as described above in the
previous examples using CD20+ cells. In addition, the selective
cytotoxicity characteristics of SLT-1A::.alpha.CD20 are determined
by the same general cell-kill assay using CD20- cells as a
comparison to the CD20+ cells. The CD.sub.50 of the cytotoxic
protein of this example is approximately 0.01-100 nM for CD20+
cells depending on the cell line. The CD.sub.50 of the cytotoxic
protein is approximately 10-10,000 fold greater (less cytotoxic)
for cells not expressing CD20 on a cellular surface as compared to
cells which do express CD20 on a cellular surface. In addition, the
cytotoxicity of SLT-1A::.alpha.CD20 is investigated for both direct
cytotoxicity and indirect cytotoxicity by T-cell epitope delivery
and presentation leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
SLT-1A::.alpha.CD20 Using Animal Models
[0521] Animal models are used to determine the in vivo effects of
the cytotoxic protein SLT-1A::.alpha.CD20 on neoplastic cells.
Various mice strains are used to test the effect of the cytotoxic
protein after intravenous administration on xenograft tumors in
mice resulting from the injection into those mice of human
neoplastic cells which express CD20 on their cell surfaces. Cell
killing is investigated for both direct cytotoxicity and indirect
cytotoxicity by T-cell epitope delivery and presentation leading to
CTL-mediated cytotoxicity.
Example 7
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Specific to HER2 (".alpha.HER2-V.sub.HH Fused
with SLT-1A")
[0522] In this example, the CD8+ T-cell hyper-immunized and
B-cell/CD4+ T-cell de-immunized Shiga toxin effector region is
derived from the A subunit of Shiga-like Toxin 1 (SLT-1A) as
described above. The immunoglobulin-type binding region is
.alpha.HER2 V.sub.HH derived from a single-domain variable region
of the camelid antibody (V.sub.HH) protein 5F7, as described in
U.S. Patent Application Publication 2011/0059090.
Construction, Production, and Purification of the Cytotoxic Protein
".alpha.HER2-V.sub.HH Fused with SLT-1A"
[0523] The immunoglobulin-type binding region and Shiga toxin
effector region are linked together to form a fused protein (see,
e.g., SEQ ID NOs: 52, 53, and 54). In this example, a
polynucleotide encoding the .alpha.HER2-V.sub.HH variable region
derived from protein 5F7 may be cloned in frame with a
polynucleotide encoding a linker known in the art and in frame with
a polynucleotide encoding the Shiga toxin effector region
comprising amino acids of SEQ ID NOs: 11-43. Variants of
".alpha.HER2-V.sub.HH fused with SLT-1A" cytotoxic proteins are
created such that the binding region is optionally located adjacent
to the amino-terminus of the Shiga toxin effector region and
optionally comprises a carboxy-terminal endoplasmic reticulum
signal motif of the KDEL family. Expression of the
".alpha.HER2-V.sub.HH fused with SLT-1A" cytotoxic protein variants
is accomplished using either bacterial and/or cell-free, protein
translation systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
".alpha.HER2-V.sub.HH Fused with SLT-1A"
[0524] The binding characteristics of the cytotoxic protein of this
example for HER2+ cells and HER2- cells is determined by a
fluorescence-based, flow-cytometry. The B.sub.max for
".alpha.HER2-V.sub.HH fused with SLT-1A" variants to HER2+ cells is
measured to be approximately 50,000-200,000 MFI with a K.sub.D
within the range of 0.01-100 nM, whereas there is no significant
binding to HER2- cells in this assay.
[0525] The ribosome inactivation abilities of the
".alpha.HER2-V.sub.HH fused with SLT-1A" cytotoxic proteins are
determined in a cell-free, in vitro protein translation as
described above in the previous examples. The inhibitory effect of
the cytotoxic protein of this example on cell-free protein
synthesis is significant. The IC.sub.50 of ".alpha.HER2-V.sub.HH
fused with SLT-1A" variants on protein synthesis in this cell-free
assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
".alpha.HER2-V.sub.HH Fused with SLT-1A" Using a HER2+ Cell-Kill
Assay
[0526] The cytotoxicity characteristics of ".alpha.HER2-V.sub.HH
fused with SLT-1A" variants are determined by the general cell-kill
assay as described above in the previous examples using HER2+
cells. In addition, the selective cytotoxicity characteristics of
".alpha.HER2-V.sub.HH fused with SLT-1A" are determined by the same
general cell-kill assay using HER2- cells as a comparison to the
HER2+ cells. The CD.sub.50 of the cytotoxic protein of this example
is approximately 0.01-100 nM for HER2+ cells depending on the cell
line. The CD.sub.50 of the cytotoxic protein is approximately
10-10,000 fold greater (less cytotoxic) for cells not expressing
HER2 on a cellular surface as compared to cells which do express
HER2 on a cellular surface. In addition, the cytotoxicity of
.alpha.HER2-V.sub.HH fused with SLT-1A is investigated for both
direct cytotoxicity and indirect cytotoxicity by T-cell epitope
delivery and presentation leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
.alpha.HER2-V.sub.HH Fused with SLT-1A Using Animal Models
[0527] Animal models are used to determine the in vivo effects of
the cytotoxic protein .alpha.HER2-V.sub.HH fused with SLT-1A on
neoplastic cells. Various mice strains are used to test the effect
of the cytotoxic protein after intravenous administration on
xenograft tumors in mice resulting from the injection into those
mice of human neoplastic cells which express HER2 on their cell
surfaces. Cell killing is investigated for both direct cytotoxicity
and indirect cytotoxicity by T-cell epitope delivery and
presentation leading to CTL-mediated cytotoxicity.
Example 8
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from the Antibody
.alpha.Epstein-Barr-Antigen
[0528] In this example, the CD8+ T-cell hyper-immunized and
B-cell/CD4+ T-cell de-immunized Shiga toxin effector region is a
de-immunized Shiga toxin effector polypeptide derived from the A
subunit of Shiga-like Toxin 1 (SLT-1A) as described above. An
immunoglobulin-type binding region aEpstein-Barr-antigen is derived
from a monoclonal antibody against an Epstein Barr antigen (Fang C
et al., J Immunol Methods 287: 21-30 (2004)), which comprises an
immunoglobulin-type binding region capable of binding a human cell
infected by the Epstein-Barr virus or a transformed cell expressing
an Epstein-Barr antigen. The Epstein-Barr antigen is expressed on
multiple cell types, such as cells infected by an Epstein-Barr
virus and cancer cells (e.g. lymphoma and nasphamygeal cancer
cells). In addition, Epstein-Barr infection is associated with
other diseases, e.g., multiple sclerosis.
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.EpsteinBarr::KDEL
[0529] The immunoglobulin-type binding region aEpstein-Barr-antigen
and Shiga toxin effector region are linked together, and a
carboxy-terminal KDEL is added to form a protein. For example, a
fusion protein is produced by expressing a polynucleotide encoding
the aEpstein-Barr-antigen-binding protein
SLT-1A::.alpha.EpsteinBarr::KDEL. Expression of the
SLT-1A::.alpha.EpsteinBarr::KDEL cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.EpsteinBarr::KDEL
[0530] The binding characteristics of the cytotoxic protein of this
example for Epstein-Barr antigen positive cells and Epstein-Barr
antigen negative cells is determined by fluorescence-based,
flow-cytometry. The B.sub.max for SLT-1A::.alpha.EpsteinBarr::KDEL
to Epstein-Barr antigen positive cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to
Epstein-Barr antigen negative cells in this assay.
[0531] The ribosome inactivation abilities of the
SLT-1A::.alpha.EpsteinBarr::KDEL cytotoxic protein is determined in
a cell-free, in vitro protein translation as described above in the
previous examples. The inhibitory effect of the cytotoxic protein
of this example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.EpsteinBarr::KDEL on protein synthesis
in this cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.EpsteinBarr::KDEL Using a Cell-Kill Assay
[0532] The cytotoxicity characteristics of
SLT-1A::.alpha.EpsteinBarr::KDEL are determined by the general
cell-kill assay as described above in the previous examples using
Epstein-Barr antigen positive cells. In addition, the selective
cytotoxicity characteristics of SLT-1A::.alpha.EpsteinBarr::KDEL
are determined by the same general cell-kill assay using
Epstein-Barr antigen negative cells as a comparison to the
Epstein-Barr antigen positive cells. The CD.sub.50 of the cytotoxic
protein of this example is approximately 0.01-100 nM for
Epstein-Barr antigen positive cells depending on the cell line. The
CD.sub.50 of the cytotoxic protein is approximately 10-10,000 fold
greater (less cytotoxic) for cells not expressing the Epstein-Barr
antigen on a cellular surface as compared to cells which do express
the Epstein-Barr antigen on a cellular surface. In addition, the
cytotoxicity of SLT-1A::.alpha.EpsteinBarr::KDEL is investigated
for both direct cytotoxicity and indirect cytotoxicity by T-cell
epitope delivery and presentation leading to CTL-mediated
cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
SLT-1A::.alpha.EpsteinBarr::KDEL Using Animal Models
[0533] Animal models are used to determine the in vivo effects of
the cytotoxic protein SLT-1A::.alpha.EpsteinBarr::KDEL on
neoplastic cells. Various mice strains are used to test the effect
of the cytotoxic protein after intravenous administration on
xenograft tumors in mice resulting from the injection into those
mice of human neoplastic cells which express Epstein-Barr antigens
on their cell surfaces. Cell killing is investigated for both
direct cytotoxicity and indirect cytotoxicity by T-cell epitope
delivery and presentation leading to CTL-mediated cytotoxicity.
Example 9
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from the Antibody
.alpha.Leishmania-Antigen
[0534] In this example, the Shiga toxin effector region is a CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga-like
Toxin 1 (SLT-1A) as described above. An immunoglobulin-type binding
region aLeishmania-antigen is derived from an antibody generated,
using techniques known in the art, to a cell-surface Leishmania
antigen present on human cells harboring an intracellular
trypanosomatid protozoa (see Silveira T et al., Int J Parasitol 31:
1451-8 (2001); Kenner J et al., J Cutan Pathol 26: 130-6 (1999);
Berman J and Dwyer, Clin Exp Immunol 44: 342-348 (1981)).
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.Leishmania::KDEL
[0535] The immunoglobulin-type binding region
.alpha.-Leishmania-antigen and Shiga toxin effector region are
linked together, and a carboxy-terminal KDEL is added to form a
protein. For example, a fusion protein is produced by expressing a
polynucleotide encoding the Leishmania-antigen-binding protein
SLT-1A::.alpha.Leishmania::KDEL. Expression of the
SLT-1A::.alpha.Leishmania::KDEL cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.Leishmania::KDEL
[0536] The binding characteristics of the cytotoxic protein of this
example for Leishmania antigen positive cells and Leishmania
antigen negative cells is determined by fluorescence-based,
flow-cytometry. The B.sub.max for SLT-1A::.alpha.Leishmania::KDEL
to Leishmania antigen positive cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to Leishmania
antigen negative cells in this assay.
[0537] The ribosome inactivation abilities of the
SLT-1A::.alpha.Leishmania::KDEL cytotoxic protein is determined in
a cell-free, in vitro protein translation as described above in the
previous examples. The inhibitory effect of the cytotoxic protein
of this example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.Leishmania::KDEL on protein synthesis
in this cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.Leishmania::KDEL Using a Cell-Kill Assay
[0538] The cytotoxicity characteristics of
SLT-1A::.alpha.Leishmania::KDEL are determined by the general
cell-kill assay as described above in the previous examples using
Leishmania antigen positive cells. In addition, the selective
cytotoxicity characteristics of SLT-1A::.alpha.Leishmania::KDEL are
determined by the same general cell-kill assay using Leishmania
antigen negative cells as a comparison to the Leishmania antigen
positive cells. The CD.sub.50 of the cytotoxic protein of this
example is approximately 0.01-100 nM for Leishmania antigen
positive cells depending on the cell line. The CD.sub.50 of the
cytotoxic protein is approximately 10-10,000 fold greater (less
cytotoxic) for cells not expressing the Leishmania antigen on a
cellular surface as compared to cells which do express the
Leishmania antigen on a cellular surface. In addition, the
cytotoxicity of SLT-1A::.alpha.Leishmania::KDEL is investigated for
both direct cytotoxicity and indirect cytotoxicity by T-cell
epitope delivery and presentation leading to CTL-mediated
cytotoxicity.
Example 10
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from an Immunoglobulin-Type Binding
Region .alpha.Neurotensin-Receptor
[0539] In this example, the Shiga toxin effector region is a CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga-like
Toxin 1 (SLT-1A) as described above. An immunoglobulin-type binding
region .alpha.Neurotensin-Receptor is derived from the DARPin.TM.
(GenBank Accession: 2P2C_R) or a monoclonal antibody (Ovigne J et
al., Neuropeptides 32: 247-56 (1998)) which binds the human
neurotensin receptor. The neurotensin receptor is expressed by
various cancer cells, such as breast cancer, colon cancer, lung
cancer, melanoma, and pancreatic cancer cells.
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.NeurotensinR::KDEL
[0540] The immunoglobulin-type binding region .alpha.NeurotensinR
and Shiga toxin effector region are linked together, and a
carboxy-terminal KDEL is added to form a protein. For example, a
fusion protein is produced by expressing a polynucleotide encoding
the neurotensin-receptor-binding protein
SLT-1A::.alpha.NeurotensinR::KDEL. Expression of the
SLT-1A::.alpha.NeurotensinR::KDEL cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.NeurotensinR::KDEL
[0541] The binding characteristics of the cytotoxic protein of this
example for neurotensin receptor positive cells and neurotensin
receptor negative cells is determined by fluorescence-based,
flow-cytometry. The B.sub.max for SLT-1A::.alpha.NeurotensinR::KDEL
to neurotensin receptor positive cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to neurotensin
receptor negative cells in this assay.
[0542] The ribosome inactivation abilities of the
SLT-1A::.alpha.NeurotensinR::KDEL cytotoxic protein is determined
in a cell-free, in vitro protein translation as described above in
the previous examples. The inhibitory effect of the cytotoxic
protein of this example on cell-free protein synthesis is
significant. The IC.sub.50 of SLT-1A::.alpha.NeurotensinR::KDEL on
protein synthesis in this cell-free assay is approximately 0.1-100
pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.NeurotensinR::KDEL Using a Cell-Kill Assay
[0543] The cytotoxicity characteristics of
SLT-1A::.alpha.NeurotensinR::KDEL are determined by the general
cell-kill assay as described above in the previous examples using
neurotensin receptor positive cells. In addition, the selective
cytotoxicity characteristics of SLT-1A::.alpha.NeurotensinR::KDEL
are determined by the same general cell-kill assay using
neurotensin receptor negative cells as a comparison to the
neurotensin receptor positive cells. The CD.sub.50 of the cytotoxic
protein of this example is approximately 0.01-100 nM for
neurotensin receptor positive cells depending on the cell line. The
CD.sub.50 of the cytotoxic protein is approximately 10-10,000 fold
greater (less cytotoxic) for cells not expressing neurotensin
receptor on a cellular surface as compared to cells which do
express neurotensin receptor on a cellular surface. In addition,
the cytotoxicity of SLT-1A::.alpha.NeurotensinR::KDEL is
investigated for both direct cytotoxicity and indirect cytotoxicity
by T-cell epitope delivery and presentation leading to CTL-mediated
cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
SLT-1A::.alpha.NeurotensinR::KDEL Using Animal Models
[0544] Animal models are used to determine the in vivo effects of
the cytotoxic protein SLT-1A::.alpha.NeurotensinR::KDEL on
neoplastic cells. Various mice strains are used to test the effect
of the cytotoxic protein after intravenous administration on
xenograft tumors in mice resulting from the injection into those
mice of human neoplastic cells which express neurotensin receptors
on their cell surfaces. Cell killing is investigated for both
direct cytotoxicity and indirect cytotoxicity by T-cell epitope
delivery and presentation leading to CTL-mediated cytotoxicity.
Example 11
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from an Immunoglobulin-Type Binding
Region .alpha.EGFR
[0545] In this example, the Shiga toxin effector region is CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga-like
Toxin 1 (SLT-1A). The binding region .alpha.EGFR is derived from
the AdNectin.TM. (GenBank Accession: 3QWQ_B), the Affibody.TM.
(GenBank Accession: 2KZI_A; U.S. Pat. No. 8,598,113), or an
antibody, all of which bind one or more human epidermal growth
factor receptors. The expression of epidermal growth factor
receptors are associated with human cancer cells, such as, e.g.,
lung cancer cells, breast cancer cells, and colon cancer cells.
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.EGFR::KDEL
[0546] The immunoglobulin-type binding region .alpha.EGFR and Shiga
toxin effector region are linked together, and a carboxy-terminal
KDEL is added to form a protein. For example, a fusion protein is
produced by expressing a polynucleotide encoding the EGFR binding
protein SLT-1A::.alpha.EGFR::KDEL. Expression of the
SLT-1A::.alpha.EGFR::KDEL cytotoxic protein is accomplished using
either bacterial and/or cell-free, protein translation systems as
described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.EGFR::KDEL
[0547] The binding characteristics of the cytotoxic protein of this
example for EGFR+ cells and EGFR- cells is determined by
fluorescence-based, flow-cytometry. The B.sub.max for
SLT-1A::.alpha.EGFR::KDEL to EGFR+ cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to EGFR- cells
in this assay.
[0548] The ribosome inactivation abilities of the
SLT-1A::.alpha.EGFR::KDEL cytotoxic protein is determined in a
cell-free, in vitro protein translation as described above in the
previous examples. The inhibitory effect of the cytotoxic protein
of this example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.EGFR::KDEL on protein synthesis in this
cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.EGFR::KDEL Using a Cell-Kill Assay
[0549] The cytotoxicity characteristics of
SLT-1A::.alpha.EGFR::KDEL are determined by the general cell-kill
assay as described above in the previous examples using EGFR+
cells. In addition, the selective cytotoxicity characteristics of
SLT-1A::.alpha.EGFR::KDEL are determined by the same general
cell-kill assay using EGFR- cells as a comparison to the Leishmania
antigen positive cells. The CD.sub.50 of the cytotoxic protein of
this example is approximately 0.01-100 nM for EGFR+ cells depending
on the cell line. The CD.sub.50 of the cytotoxic protein is
approximately 10-10,000 fold greater (less cytotoxic) for cells not
expressing EGFR on a cellular surface as compared to cells which do
express EGFR on a cellular surface. In addition, the cytotoxicity
of SLT-1A::.alpha.EGFR::KDEL is investigated for both direct
cytotoxicity and indirect cytotoxicity by T-cell epitope delivery
and presentation leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
SLT-1A::.alpha.EGFR::KDEL Using Animal Models
[0550] Animal models are used to determine the in vivo effects of
the cytotoxic protein SLT-1A::.alpha.EGFR::KDEL on neoplastic
cells. Various mice strains are used to test the effect of the
cytotoxic protein after intravenous administration on xenograft
tumors in mice resulting from the injection into those mice of
human neoplastic cells which express EGFR(s) on their cell
surfaces. Cell killing is investigated for both direct cytotoxicity
and indirect cytotoxicity by T-cell epitope delivery and
presentation leading to CTL-mediated cytotoxicity.
Example 12
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from the Antibody .alpha.CCR5
[0551] In this example, the Shiga toxin effector region is a CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga-like
Toxin 1 (SLT-1A). An immunoglobulin-type binding region .alpha.CCR5
is derived from a monoclonal antibody against human CCR5 (CD195)
(Bernstone L et al., Hybridoma 31: 7-19 (2012)). CCR5 is
predominantly expressed on T-cells, macrophages, dendritic cells,
and microglia. In addition, CCR5 plays a role in the pathogenesis
and spread of the Human Immunodeficiency Virus (HIV).
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.CCR5::KDEL
[0552] The immunoglobulin-type binding region .alpha.CCR5 and Shiga
toxin effector region are linked together, and a carboxy-terminal
KDEL is added to form a protein. For example, a fusion protein is
produced by expressing a polynucleotide encoding the
.alpha.CCR5-binding protein SLT-1A::.alpha.CCR5::KDEL. Expression
of the SLT-1A::.alpha.CCR5::KDEL cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.CCR5
[0553] The binding characteristics of the cytotoxic protein of this
example for CCR5+ cells and CCR5- cells is determined by
fluorescence-based, flow-cytometry. The B.sub.max for
SLT-1A::.alpha.CCR5::KDEL to CCR5+ positive cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to CCR5- cells
in this assay.
[0554] The ribosome inactivation abilities of the
SLT-1A::.alpha.CCR5::KDEL cytotoxic protein is determined in a
cell-free, in vitro protein translation as described above in the
previous examples. The inhibitory effect of the cytotoxic protein
of this example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.CCR5::KDEL on protein synthesis in this
cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.CCR5::KDEL Using a Cell-Kill Assay
[0555] The cytotoxicity characteristics of
SLT-1A::.alpha.CCR5::KDEL are determined by the general cell-kill
assay as described above in the previous examples using CCR5+
cells. In addition, the selective cytotoxicity characteristics of
SLT-1A::.alpha.CCR5::KDEL are determined by the same general
cell-kill assay using CCR5- cells as a comparison to the CCR5+
cells. The CD.sub.50 of the cytotoxic protein of this example is
approximately 0.01-100 nM for CCR5+ cells depending on the cell
line. The CD.sub.50 of the cytotoxic protein is approximately
10-10,000 fold greater (less cytotoxic) for cells not expressing
CCR5 on a cellular surface as compared to cells which do express
CCR5 on a cellular surface. In addition, the cytotoxicity of
SLT-1A::.alpha.CCR5::KDEL is investigated for both direct
cytotoxicity and indirect cytotoxicity by T-cell epitope delivery
and presentation leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
SLT-1A::.alpha.CCR5::KDEL Using Animal Models
[0556] Animal models are used to determine the in vivo effects of
the cytotoxic protein SLT-1A::.alpha.CCR5::KDEL on depleting
T-cells from donor materials (see Tsirigotis P et al.,
Immunotherapy 4: 407-24 (2012)). Non-human primates are used to
determine in vivo effects of SLT-1A::.alpha.CCR5. Graft versus host
disease is analyzed in rhesus macaques after kidney transplantation
when the donated organs are pretreated with
SLT-1A::.alpha.CCR5::KDEL (see Weaver T et al., Nat Med 15: 746-9
(2009)). In vivo depletion of peripheral blood T lymphocytes in
cynomolgus primates is observed after parenteral administration of
different doses of SLT-1A::.alpha.CCR5::KDEL. Cell killing is
investigated for both direct cytotoxicity and indirect cytotoxicity
by T-cell epitope delivery and presentation leading to CTL-mediated
cytotoxicity. The use of SLT-1A::.alpha.CCR5::KDEL to block HIV
infection is tested by giving an acute dose of
SLT-1A::.alpha.CCR5::KDEL to non-human primates in order to
severely deplete circulating T-cells upon exposure to a simian
immunodeficiency virus (SIV) (see Sellier P et al., PLoS One 5:
e10570 (2010)).
Example 13
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from an Anti-Env Immunoglobulin
Domain
[0557] In this example, the Shiga toxin effector region is a CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga
toxin (StxA). An immunoglobulin-type binding region .alpha.Env is
derived from existing antibodies that bind HIV envelope
glycoprotein (Env), such as GP41, GP120, GP140, or GP160 (see e.g.
Chen W et al., J Mol Bio 382: 779-89 (2008); Chen W et al., Expert
Opin Biol Ther 13: 657-71 (2013); van den Kerkhof T et al.,
Retrovirology 10: 102 (2013)) or from antibodies generated using
standard techniques (see Prabakaran et al., Front Microbiol 3: 277
(2012)). Envs are HIV surface proteins that are also displayed on
the cell surfaces of HIV-infected cells during HIV replication.
Although Envs are expressed in infected cells predominantly in
endosomal compartments, sufficient amounts of Envs could be present
on a cell surface to be targeted by a highly potent cytotoxic,
cell-targeted protein of the invention. In addition, Env-targeting
cytotoxic proteins might bind HIV virions and enter newly infected
cells during the fusion of virions with a host cell.
[0558] Because HIV displays a high rate of mutation, it is
preferable to use an immunoglobulin domain that binds a functional
constrained part of an Env, such as shown by broadly neutralizing
antibodies that bind Envs from multiple strains of HIV (van den
Kerkhof T et al., Retrovirology 10: 102 (2013)). Because the Envs
present on an infected cell's surface are believed to present
sterically restricted epitopes (Chen W et al., J Virol 88: 1125-39
(2014)), it is preferable to use smaller than 100 kD and ideally
smaller than 25 kD, such as sdAbs or V.sub.HH domains.
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.Env::KDEL
[0559] The immunoglobulin-type binding region .alpha.Env and Shiga
toxin effector region are linked together, and a carboxy-terminal
KDEL is added to form a cytotoxic protein. For example, a fusion
protein is produced by expressing a polynucleotide encoding the
.alpha.Env-binding protein SLT-1A::.alpha.Env::KDEL. Expression of
the SLT-1A::.alpha.Env::KDEL cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.Env::KDEL
[0560] The binding characteristics of the cytotoxic protein of this
example for Env+ cells and Env- cells is determined by
fluorescence-based, flow-cytometry assay. The B.sub.max for
SLT-1A::.alpha.Env::KDEL to Env+ positive cells is measured to be
approximately 50,000-200,000 MFI with a K.sub.D within the range of
0.01-100 nM, whereas there is no significant binding to Env- cells
in this assay.
[0561] The ribosome inactivation abilities of the
SLT-1A::.alpha.Env::KDEL cytotoxic protein is determined in a
cell-free, in vitro protein translation as described above in the
previous examples. The inhibitory effect of the cytotoxic protein
of this example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.Env::KDEL on protein synthesis in this
cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.Env::KDEL Using a Cell-Kill Assay
[0562] The cytotoxicity characteristics of SLT-1A::.alpha.Env::KDEL
are determined by the general cell-kill assay as described above in
the previous examples using Env+ cells. In addition, the selective
cytotoxicity characteristics of SLT-1A::.alpha.Env::KDEL are
determined by the same general cell-kill assay using Env-cells as a
comparison to the Env+ cells. The CD.sub.50 of the cytotoxic
protein of this example is approximately 0.01-100 nM for Env+ cells
depending on the cell line and/or the HIV strain used to infect the
cells to make them Env+. The CD.sub.50 of the cytotoxic protein is
approximately 10-10,000 fold greater (less cytotoxic) for cells not
expressing Env on a cellular surface as compared to cells which do
express Env on a cellular surface. In addition, the cytotoxicity of
SLT-1A::.alpha.Env::KDEL is investigated for both direct
cytotoxicity and indirect cytotoxicity by T-cell epitope delivery
and presentation leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein SLT-1A::
Env::KDEL Using Animal Models
[0563] The use of SLT-1A::.alpha.Env::KDEL to inhibit HIV infection
is tested by administering SLT-1A::.alpha.Env::KDEL to simian
immunodeficiency virus (SIV) infected non-human primates (see
Sellier P et al., PLoS One 5: e10570 (2010)). Cell killing is
investigated for both direct cytotoxicity and indirect cytotoxicity
by T-cell epitope delivery and presentation leading to CTL-mediated
cytotoxicity.
Example 14
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide
and a Binding Region Derived from the Antibody .alpha.UL18
[0564] In this example, the Shiga toxin effector region is a CD8+
T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga-like
Toxin 1 (SLT-1A). An immunoglobulin-type binding region .alpha.UL18
is derived from an antibody generated, using techniques known in
the art, to the cell-surface cytomegalovirus protein UL18, which is
present on human cells infected with cytomegalovirus (Yang Z,
Bjorkman P, Proc Natl Acad Sci USA 105: 10095-100 (2008)). The
human cytomegalovirus infection is associated with various cancers
and inflammatory disorders.
Construction, Production, and Purification of the Cytotoxic Protein
SLT-1A::.alpha.UL18::KDEL
[0565] The immunoglobulin-type binding region .alpha.UL18 and Shiga
toxin effector region are linked together, and a carboxy-terminal
KDEL is added to form a protein. For example, a fusion protein is
produced by expressing a polynucleotide encoding the
.alpha.UL18-binding protein SLT-1A::.alpha.UL18::KDEL. Expression
of the SLT-1A::.alpha.UL18::KDEL cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
SLT-1A::.alpha.UL18::KDEL
[0566] The binding characteristics of the cytotoxic protein of this
example for cytomegalovirus protein UL18 positive cells and
cytomegalovirus protein UL18 negative cells is determined by
fluorescence-based, flow-cytometry. The B.sub.max for
SLT-1A::.alpha.UL18::KDEL to cytomegalovirus protein UL18 positive
cells is measured to be approximately 50,000-200,000 MFI with a
K.sub.D within the range of 0.01-100 nM, whereas there is no
significant binding to cytomegalovirus protein UL18 negative cells
in this assay.
[0567] The ribosome inactivation abilities of the
SLT-1A::.alpha.UL18::KDEL cytotoxic protein is determined in a
cell-free, in vitro protein translation as described above in the
previous examples. The inhibitory effect of the cytotoxic protein
of this example on cell-free protein synthesis is significant. The
IC.sub.50 of SLT-1A::.alpha.UL18::KDEL on protein synthesis in this
cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
SLT-1A::.alpha.UL18::KDEL Using a Cell-Kill Assay
[0568] The cytotoxicity characteristics of
SLT-1A::.alpha.UL18::KDEL are determined by the general cell-kill
assay as described above in the previous examples using
cytomegalovirus protein UL18 positive cells. In addition, the
selective cytotoxicity characteristics of SLT-1A::.alpha.UL18::KDEL
are determined by the same general cell-kill assay using
cytomegalovirus protein UL18 negative cells as a comparison to the
cytomegalovirus protein UL18 positive cells. The CD.sub.50 of the
cytotoxic protein of this example is approximately 0.01-100 nM for
cytomegalovirus protein UL18 positive cells depending on the cell
line. The CD.sub.50 of the cytotoxic protein is approximately
10-10,000 fold greater (less cytotoxic) for cells not expressing
the cytomegalovirus protein UL18 on a cellular surface as compared
to cells which do express the cytomegalovirus protein UL18 on a
cellular surface. In addition, the cytotoxicity of
SLT-1A::.alpha.UL18::KDEL is investigated for both direct
cytotoxicity and indirect cytotoxicity by T-cell epitope delivery
and presentation leading to CTL-mediated cytotoxicity.
Example 15
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Diphtheria Toxin Effector
Polypeptide and a Binding Region Specific to CD20 (.alpha.CD20
Fused with Diphtheria Toxin)
[0569] In this example, a CD8+ T-cell hyper-immunized and
B-cell/CD4+ T-cell de-immunized diphtheria toxin effector region is
derived from the A subunit of diphtheria toxin 1 as described
above. An immunoglobulin-type binding region .alpha.CD20-antigen is
derived from an immunoglobulin-type domain recognizing human CD20
(see e.g. Haisma et al., Blood 92: 184-90 (1999); Geng S et al.,
Cell Mol Immunol 3: 439-43 (2006); Olafesn T et al., Protein Eng
Des Sel 23: 243-9 (2010)), which comprises an immunoglobulin-type
binding region capable of binding an extracellular part of CD20.
CD20 is expressed on multiple cancer cell types, such as, e.g.,
B-cell lymphoma cells, hairy cell leukemia cells, B-cell chronic
lymphocytic leukemia cells, and melanoma cells. In addition, CD20
is an attractive target for therapeutics to treat certain
autoimmune diseases, disorders, and conditions involving overactive
B-cells.
Construction, Production, and Purification of the Cytotoxic Protein
Diphtheria Toxin::.alpha.CD20
[0570] The immunoglobulin-type binding region .alpha.CD20 and
diphtheria toxin effector region (such as, e.g., SEQ ID NOs: 46,
47, and 48) are linked together. For example, a fusion protein is
produced by expressing a polynucleotide encoding the
.alpha.CD20-antigen-binding protein diphtheria toxin::.alpha.CD20
(see, e.g., SEQ ID NOs: 55, 56, and 57). Expression of the SLT
diphtheria toxin::.alpha.CD20 cytotoxic protein is accomplished
using either bacterial and/or cell-free, protein translation
systems as described in the previous examples.
Determining the In Vitro Characteristics of the Cytotoxic Protein
Diphtheria Toxin::.alpha.CD20
[0571] The binding characteristics of the cytotoxic protein of this
example for CD20+ cells and CD20- cells is determined by
fluorescence-based, flow-cytometry assay as described in previous
patents. The B.sub.max for diphtheria toxin::.alpha.CD20 to CD20+
cells is measured to be approximately 50,000-200,000 MFI with a
K.sub.D within the range of 0.01-100 nM, whereas there is no
significant binding to CD20- cells in this assay.
[0572] The ribosome inactivation abilities of the diphtheria
toxin::.alpha.CD20 cytotoxic protein is determined in a cell-free,
in vitro protein translation as described above in the previous
examples. The inhibitory effect of the cytotoxic protein of this
example on cell-free protein synthesis is significant. The
IC.sub.50 of diphtheria toxin::.alpha.CD20 on protein synthesis in
this cell-free assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein Diphtheria
Toxin::.alpha.CD20 Using a CD20+ Cell-Kill Assay
[0573] The cytotoxicity characteristics of diphtheria
toxin::.alpha.CD20 are determined by the general cell-kill assay as
described above in the previous examples using CD20+ cells. In
addition, the selective cytotoxicity characteristics of diphtheria
toxin::.alpha.CD20 are determined by the same general cell-kill
assay using CD20- cells as a comparison to the CD20+ cells. The
CD.sub.50 of the cytotoxic protein of this example is approximately
0.01-100 nM for CD20+ cells depending on the cell line. The
CD.sub.50 of the cytotoxic protein is approximately 10-10,000 fold
greater (less cytotoxic) for cells not expressing CD20 on a
cellular surface as compared to cells which do express CD20 on a
cellular surface. In addition, the cytotoxicity of diphtheria
toxin::.alpha.CD20 is investigated for both direct cytotoxicity and
indirect cytotoxicity by T-cell epitope delivery and presentation
leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein Diphtheria
Toxin::.alpha.CD20 Using Animal Models
[0574] Animal models are used to determine the in vivo effects of
the cytotoxic protein diphtheria toxin::.alpha.CD20 on neoplastic
cells. Various mice strains are used to test the effect of the
cytotoxic protein after intravenous administration on xenograft
tumors in mice resulting from the injection into those mice of
human neoplastic cells which express CD20 on their cell surfaces.
Cell killing is investigated for both direct cytotoxicity and
indirect cytotoxicity by T-cell epitope delivery and presentation
leading to CTL-mediated cytotoxicity.
Example 16
A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized and
B-Cell/CD4+ T-Cell De-Immunized Diphtheria Toxin Effector
Polypeptide and a Binding Region Specific to HER2
(".alpha.HER2-V.sub.HH Fused with Diphtheria Toxin")
[0575] In this example, the CD8+ T-cell hyper-immunized and
B-cell/CD4+ T-cell de-immunized diphtheria toxin effector region is
derived from the A subunit of diphtheria toxin as described above.
The immunoglobulin-type binding region is .alpha.HER2 V.sub.HH
derived from a single-domain variable region of the camelid
antibody (V.sub.HH) protein 5F7, as described in U.S. Patent
Application Publication 2011/0059090.
Construction, Production, and Purification of the Cytotoxic Protein
".alpha.HER2-V.sub.HH Fused with Diphtheria Toxin"
[0576] The immunoglobulin-type binding region and diphtheria toxin
effector region are linked together to form a fused protein (see,
e.g., SEQ ID NOs: 58, 59, and 60). In this example, a
polynucleotide encoding the .alpha.HER2-V.sub.HH variable region
derived from protein 5F7 may be cloned in frame with a
polynucleotide encoding a linker known in the art and in frame with
a polynucleotide encoding the diphtheria toxin effector region
comprising amino acids of SEQ ID NOs: 46, 47, or 48. Variants of
".alpha.HER2-V.sub.HH fused with diphtheria toxin" cytotoxic
proteins are created such that the binding region is optionally
located adjacent to the amino-terminus of the diphtheria toxin
effector region and optionally comprises a carboxy-terminal
endoplasmic reticulum signal motif of the KDEL family. Expression
of the ".alpha.HER2-V.sub.HH fused with diphtheria toxin" cytotoxic
protein variants is accomplished using either bacterial and/or
cell-free, protein translation systems as described in the previous
examples.
Determining the In Vitro Characteristics of the Cytotoxic Proteins
".alpha.HER2-V.sub.HH Fused with Diphtheria Toxin"
[0577] The binding characteristics of the cytotoxic protein of this
example for HER2+ cells and HER2- cells is determined by
fluorescence-based, flow-cytometry assay as described in previous
patents. The B.sub.max for ".alpha.HER2-V.sub.HH fused with
diphtheria toxin" to HER2+ cells is measured to be approximately
50,000-200,000 MFI with a K.sub.D within the range of 0.01-100 nM,
whereas there is no significant binding to HER2- cells in this
assay.
[0578] The ribosome inactivation abilities of the
".alpha.HER2-V.sub.HH fused with diphtheria toxin" cytotoxic
proteins is determined in a cell-free, in vitro protein translation
as described above in the previous examples. The inhibitory effect
of the cytotoxic protein of this example on cell-free protein
synthesis is significant. The IC.sub.50 of ".alpha.HER2-V.sub.HH
fused with diphtheria toxin" on protein synthesis in this cell-free
assay is approximately 0.1-100 pM.
Determining the Cytotoxicity of the Cytotoxic Protein
".alpha.HER2-V.sub.HH Fused with Diphtheria Toxin" Using a HER2+
Cell-Kill Assay
[0579] The cytotoxicity characteristics of ".alpha.HER2-V.sub.HH
fused with diphtheria toxin" are determined by the general
cell-kill assay as described above in the previous examples using
HER2+ cells. In addition, the selective cytotoxicity
characteristics of ".alpha.HER2-V.sub.HH fused with diphtheria
toxin" are determined by the same general cell-kill assay using
HER2- cells as a comparison to the HER2+ cells. The CD.sub.50 of
the cytotoxic protein of this example is approximately 0.01-100 nM
for HER2+ cells depending on the cell line. The CD.sub.50 of the
cytotoxic protein is approximately 10-10,000 fold greater (less
cytotoxic) for cells not expressing HER2 on a cellular surface as
compared to cells which do express HER2 on a cellular surface. In
addition, the cytotoxicity of ".alpha.HER2-V.sub.HH fused with
diphtheria toxin" is investigated for both direct cytotoxicity and
indirect cytotoxicity by T-cell epitope delivery and presentation
leading to CTL-mediated cytotoxicity.
Determining the In Vivo Effects of the Cytotoxic Protein
".alpha.HER2-V.sub.HH Fused with Diphtheria Toxin" Using Animal
Models
[0580] Animal models are used to determine the in vivo effects of
the cytotoxic protein ".alpha.HER2-V.sub.HH fused with diphtheria
toxin" on neoplastic cells. Various mice strains are used to test
the effect of the cytotoxic protein after intravenous
administration on xenograft tumors in mice resulting from the
injection into those mice of human neoplastic cells which express
HER2 on their cell surfaces. Cell killing is investigated for both
direct cytotoxicity and indirect cytotoxicity by T-cell epitope
delivery and presentation leading to CTL-mediated cytotoxicity.
Example 17
T-Cell Hyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized Shiga
Toxin Derived Cytotoxic Proteins Targeting Various Cell Types
[0581] In this example, the Shiga toxin effector region comprises
T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga
toxin effector polypeptide derived from the A subunit of Shiga-like
Toxin 1 (SLT-1A), Shiga toxin (StxA), and/or Shiga-like Toxin 2
(SLT-2A) with any one or more of the aforementioned B-cell epitope
regions disrupted via one or more embedded or inserting T-cell
epitopes. A binding region is derived from the immunoglobulin
domain from the molecule chosen from column 1 of Table 15 and which
binds the extracellular target biomolecule indicated in column 2 of
Table 15. The exemplary cytotoxic proteins of this example are
optionally created with a carboxy-terminal KDEL-type signal motif
and/or detection promoting agent(s) using reagents and techniques
known in the art. The exemplary cytotoxic proteins of this example
are tested as described in the previous examples using cells
expressing the appropriate extracellular target biomolecules. The
exemplary proteins of this example may be used, e.g., to diagnose
and treat diseases, conditions, and/or disorders indicated in
column 3 of Table 15.
TABLE-US-00018 TABLE 15 Various Binding Regions for Cell Targeting
of Cytotoxic Proteins Source of binding region Extracellular target
Application(s) alemtuzumab CD52 B-cell cancers, such as lymphoma
and leukemia, and B-cell related immune disorders, such as
autoimmune disorders basiliximab CD25 T-cell disorders, such as
prevention of organ transplant rejections, and some B-cell lineage
cancers brentuximab CD30 hematological cancers, B-cell related
immune disorders, and T-cell related immune disorders catumaxomab
EpCAM various cancers, such as ovarian cancer, malignant ascites,
gastric cancer cetuximab EGFR various cancers, such as colorectal
cancer and head and neck cancer daclizumab CD25 B-cell lineage
cancers and T-cell disorders, such as rejection of organ
transplants daratumumab CD38 hematological cancers, B-cell related
immune disorders, and T-cell related immune disorders dinutuximab
ganglioside GD2 Various cancers, such as breast cancer, myeloid
cancers, and neuroblastoma efalizumab LFA-1 (CD11a) autoimmune
disorders, such as psoriasis ertumaxomab HER2/neu various cancers
and tumors, such as breast cancer and colorectal cancer gemtuzumab
CD33 myeloid cancer or immune disorder ibritumomab CD20 B-cell
cancers, such as lymphoma and leukemia, and B-cell related immune
disorders, such as autoimmune disorders ipilimumab CD152 T-cell
related disorders and various cancers, such as leukemia, melanoma
muromonab CD3 prevention of organ transplant rejections natalizumab
integrin .alpha.4 autoimmune disorders, such as multiple sclerosis
and Crohn's disease obinutuzumab CD20 B-cell cancers, such as
lymphoma and leukemia, and B-cell related immune disorders, such as
autoimmune disorders ocaratuzumab CD20 B-cell cancers, such as
lymphoma and leukemia, and B-cell related immune disorders, such as
autoimmune disorders ocrelizumab CD20 B-cell cancers, such as
lymphoma and leukemia, and B-cell related immune disorders, such as
autoimmune disorders ofatumumab CD20 B-cell cancers, such as
lymphoma and leukemia, and B-cell related immune disorders, such as
autoimmune disorders palivizumab F protein of respiratory treat
respiratory syncytial virus syncytial virus panitumumab EGFR
various cancers, such as colorectal cancer and head and neck cancer
pertuzumab HER2/neu various cancers and tumors, such as breast
cancer and colorectal cancer pro 140 CCR5 HIV infection and T-cell
disorders ramucirumab VEGFR2 various cancers and cancer related
disorders, such as solid tumors rituximab CD20 B-cell cancers, such
as lymphoma and leukemia, and B-cell related immune disorders, such
as autoimmune disorders tocilizumab or IL-6 receptor autoimmune
disorders, atlizumab such as rheumatoid arthritis tositumomab CD20
B-cell cancers, such as lymphoma and leukemia, and B-cell related
immune disorders, such as autoimmune disorders trastuzumab HER2/neu
various cancers and tumors, such as breast cancer and colorectal
cancer ublituximab CD20 B-cell cancers, such as lymphoma and
leukemia, and B-cell related immune disorders, such as autoimmune
disorders vedolizumab integrin .alpha.4.beta.7 autoimmune
disorders, such as Crohn's disease and ulcerative colitis CD20
binding scFv(s) CD20 B-cell cancers, such as Geng S et al., Cell
Mol lymphoma and Immunol 3: 439-43 leukemia, and B-cell (2006);
Olafesn T et related immune al., Protein Eng Des disorders, such as
Sel 23: 243-9 (2010) autoimmune disorders CD22 binding scFv(s) CD22
B-cell cancers or B-cell Kawas S et al., MAbs related immune 3:
479-86 (2011) disorders CD25 binding scFv(s) CD25 various cancers
of the Muramatsu H et al., B-cell lineage and Cancer Lett 225: 225-
immune disorders 36 (2005) related to T-cells CD30 binding CD30
B-cell cancers or B- monoclonal cell/T-cell related antibody(s)
immune disorders Klimka A et al., Br J Cancer 83: 252-60 (2000)
CD33 binding CD33 myeloid cancer or monoclonal immune disorder
antibody(s) Benedict C et al., J Immunol Methods 201: 223-31 (1997)
CD38 binding CD38 hematological cancers, immunoglobulin B-cell
related immune domains U.S. Pat No. disorders, and T-cell 8,153,765
related immune disorders CD40 binding scFv(s) CD40 various cancers
and Ellmark P et al., immune disorders Immunology 106: 456- 63
(2002) CD52 binding CD52 B-cell cancers, such as monoclonal
lymphoma and antibody(s) leukemia, and B-cell U.S. Pat. No.
7,910,104 related immune B2 disorders, such as autoimmune disorders
CD56 binding CD56 immune disorders and monoclonal various cancers,
such as antibody(s) lung cancer, Merkel cell Shin J et al.,
carcinoma, myeloma Hybridoma 18: 521-7 (1999) CD79 binding CD79
B-cell cancers or B-cell monoclonal related immune antibody(s)
disorders Zhang L et al., Ther Immunol 2: 191-202 (1995) CD248
binding CD248 various cancers, such as scFv(s) inhibiting
angiogenesis Zhao A et al., J Immunol Methods 363: 221-32 (2011)
EpCAM binding EpCAM various cancers, such as monoclonal ovarian
cancer, antibody(s) malignant ascites, Schanzer J et al., J gastric
cancer Immunother 29: 477- 88 (2006) PSMA binding PSMA prostate
cancer monoclonal antibody(s) Frigerio B et al., Eur J Cancer 49:
2223-32 (2013) Eph-B2 binding Eph-B2 for various cancers such
monoclonal as colorectal cancer and antibody(s) prostate cancer
Abengozar M et al., Blood 119: 4565-76 (2012) Endoglin binding
Endoglin various cancers, such as monoclonal breast cancer and
antibody(s) colorectal cancers Volkel T et al., Biochim Biophys Res
Acta 1663: 158-66 (2004) FAP binding FAP various cancers, such as
monoclonal sarcomas and bone antibody(s) cancers Zhang J et al.,
FASEB J 27: 581-9 (2013) CEA binding CEA various cancers, such as
antibody(s) and gastrointestinal cancer, scFv(s) pancreatic cancer,
lung Neumaier M et al., cancer, and breast Cancer Res 50: 2128-
cancer 34 (1990); Pavoni E et al., BMC Cancer 6: 4 (2006); Yazaki P
et al., Nucl Med Biol 35: 151-8 (2008); Zhao J et al., Oncol Res
17: 217-22 (2008) CD24 binding CD24 various cancers, such as
monoclonal bladder cancer antibody(s) Kristiansen G et al., Lab
Invest 90: 1102-16 (2010) LewisY antigen LewisY antigens various
cancers, such as binding scFv(s) cervical cancer and Power B et
al., Protein uterine cancer Sci 12: 734-47 (2003); monoclonal
antibody BR96 Feridani A et al., Cytometry 71: 361-70 (2007)
adalimumab TNF-.alpha. various cancers and immune disorders, such
as Rheumatoid arthritis, Crohn's Disease, Plaque Psoriasis,
Psoriatic
Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis,
Hemolytic disease of the newborn afelimomab TNF-.alpha. various
cancers and immune disorders ald518 IL-6 various cancers and immune
disorders, such as rheumatoid arthritis anrukinzumab or ima- IL-13
various cancers and 638 immune disorders briakinumab IL-12, IL-23
various cancers and immune disorders, such as psoriasis, rheumatoid
arthritis, inflammatory bowel diseases, multiple sclerosis
brodalumab IL-17 various cancers and immune disorders, such as
inflammatory diseases canakinumab IL-1 various cancers and immune
disorders, such as rheumatoid arthritis certolizumab TNF-.alpha.
various cancers and immune disorders, such as Crohn's disease
fezakinumab IL-22 various cancers and immune disorders, such as
rheumatoid arthritis, psoriasis ganitumab IGF-I various cancers
golimumab TNF-.alpha. various cancers and immune disorders, such as
rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis
infliximab TNF-.alpha. various cancers and immune disorders, such
as rheumatoid arthritis, ankylosing spondylitis, psoriatic
arthritis, psoriasis, Crohn.alpha.s disease, ulcerative colitis
ixekizumab IL-17A various cancers and immune disorders, such as
autoimmune diseases mepolizumab IL-5 various immune disorders and
cancers, such as B-cell cancers nerelimomab TNF-.alpha. various
cancers and immune disorders olokizumab IL6 various cancers and
immune disorders ozoralizumab TNF-.alpha. inflammation perakizumab
IL17A various cancers and immune disorders, such as arthritis
placulumab human TNF various immune disorders and cancers sarilumab
IL6 various cancers and immune disorders, such as rheumatoid
arthritis, ankylosing spondylitis siltuximab IL-6 various cancers
and immune disorders sirukumab IL-6 various cancers and immune
disorders, such as rheumatoid arthritis tabalumab BAFF B-cell
cancers ticilimumab or CTLA-4 various cancers tremelimumab
tildrakizumab IL23 immunologically mediated inflammatory disorders
tnx-650 IL-13 various cancers and immune disorders, such as B-cell
cancers tocilizumab or IL-6 receptor various cancers and atlizumab
immune disorders, such as rheumatoid arthritis ustekinumab IL-12,
IL-23 various cancers and immune disorders, such as multiple
sclerosis, psoriasis, psoriatic arthritis Various growth VEGFR,
EGFR, various cancer, such as factors: VEGF, EGF1, FGFR breast
cancer and colon EGF2, FGF cancer, and to inhibit vascularization
Various cytokines: IL- IL-2R, IL-6R, IL-23R, various immune 2,
IL-6, IL-23, CCL2, CD80/CD86, disorders and cancers BAFFs, TNFs,
TNFRSF13/ RANKL TNFRSF17, TNFR Broadly neutralizing Influenza
surface viral infections antibodies identified antigens, e.g. from
patient samples Prabakaran et al., hemaglutinins and Front
Microbiol 3: 277 influenza matrix (2012) protein 2 Broadly
neutralizing Coronavirus surface viral infections antibodies
identified antigens from patient samples Prabakaran et al., Front
Microbiol 3: 277 (2012) Broadly neutralizing Henipaviruses surface
viral infections antibodies identified antigens from patient
samples Prabakaran et al., Front Microbiol 3: 277 (2012)
[0582] While some embodiments of the invention have been described
by way of illustration, it will be apparent that the invention can
be put into practice with many modifications, variations and
adaptations, and with the use of numerous equivalents or
alternative solutions that are within the scope of persons skilled
in the art, without departing from the spirit of the invention or
exceeding the scope of the claims.
[0583] All publications, patents, and patent applications are
herein incorporated by reference in their entirety to the same
extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety. The disclosures of U.S.
provisional patent application Ser. Nos. 61/777,130, 61/932,000,
61/951,110, 61/951,121, 62/010,918, and 62/049,325 are each
incorporated herein by reference in their entirety. The disclosures
of U.S. patent application publications US 2007/0298434 A1, US
2009/0156417 A1, and US 2013/0196928 A1 are each incorporated here
by reference in their entirety. The disclosures of international
PCT patent application serial numbers PCT/US2014/023231 and
PCT/US2014/023198 are each incorporated herein by reference in
their entirety. The complete disclosures of all electronically
available biological sequence information from GenBank (National
Center for Biotechnology Information, U.S.) for amino acid and
nucleotide sequences cited herein are each incorporated herein by
reference in their entirety.
Sequence CWU 1
1
601293PRTEscherichia colisource/note="Shiga-like toxin 1 Subunit A
(SLT-1A)" 1Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val
Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu
Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile Asp
Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg Gly
Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu Ile
Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn Arg
Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His Val
Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105 110
Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met 115
120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met
Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala
Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu Arg
Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp Asp
Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val Asp
Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro Asp
Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser Phe
Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230 235
240 Asn Cys His His His Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu
245 250 255 Phe Pro Ser Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile
Thr His 260 265 270 Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala
Ile Leu Met Arg 275 280 285 Arg Thr Ile Ser Ser 290 2293PRTShigella
dysenteriaesource/note="Shiga toxin Subunit A (StxA)" 2Lys Glu Phe
Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu
Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25
30 Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Thr Gly Asp Asn
35 40 45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly
Arg Phe 50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu
Tyr Val Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr
Arg Phe Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr
Ala Val Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln
Arg Val Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg
His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser
Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155
160 Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg
165 170 175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr
Val Met 180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly
Arg Leu Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser
Val Arg Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile
Leu Gly Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His
Ala Ser Arg Val Ala Arg Met Ala Ser Asp Glu 245 250 255 Phe Pro Ser
Met Cys Pro Ala Asp Gly Arg Val Arg Gly Ile Thr His 260 265 270 Asn
Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu Met Arg 275 280
285 Arg Thr Ile Ser Ser 290 3297PRTEscherichia
colisource/note="Shiga-like toxin 2 Subunit A (SLT-2A)" 3Asp Glu
Phe Thr Val Asp Phe Ser Ser Gln Lys Ser Tyr Val Asp Ser 1 5 10 15
Leu Asn Ser Ile Arg Ser Ala Ile Ser Thr Pro Leu Gly Asn Ile Ser 20
25 30 Gln Gly Gly Val Ser Val Ser Val Ile Asn His Val Leu Gly Gly
Asn 35 40 45 Tyr Ile Ser Leu Asn Val Arg Gly Leu Asp Pro Tyr Ser
Glu Arg Phe 50 55 60 Asn His Leu Arg Leu Ile Met Glu Arg Asn Asn
Leu Tyr Val Ala Gly 65 70 75 80 Phe Ile Asn Thr Glu Thr Asn Ile Phe
Tyr Arg Phe Ser Asp Phe Ser 85 90 95 His Ile Ser Val Pro Asp Val
Ile Thr Val Ser Met Thr Thr Asp Ser 100 105 110 Ser Tyr Ser Ser Leu
Gln Arg Ile Ala Asp Leu Glu Arg Thr Gly Met 115 120 125 Gln Ile Gly
Arg His Ser Leu Val Gly Ser Tyr Leu Asp Leu Met Glu 130 135 140 Phe
Arg Gly Arg Ser Met Thr Arg Ala Ser Ser Arg Ala Met Leu Arg 145 150
155 160 Phe Val Thr Val Ile Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln
Arg 165 170 175 Gly Phe Arg Pro Ala Leu Ser Glu Ala Ser Pro Leu Tyr
Thr Met Thr 180 185 190 Ala Gln Asp Val Asp Leu Thr Leu Asn Trp Gly
Arg Ile Ser Asn Val 195 200 205 Leu Pro Glu Tyr Arg Gly Glu Glu Gly
Val Arg Ile Gly Arg Ile Ser 210 215 220 Phe Asn Ser Leu Ser Ala Ile
Leu Gly Ser Val Ala Val Ile Leu Asn 225 230 235 240 Cys His Ser Thr
Gly Ser Tyr Ser Val Arg Ser Val Ser Gln Lys Gln 245 250 255 Lys Thr
Glu Cys Gln Ile Val Gly Asp Arg Ala Ala Ile Lys Val Asn 260 265 270
Asn Val Leu Trp Glu Ala Asn Thr Ile Ala Ala Leu Leu Asn Arg Lys 275
280 285 Pro Gln Asp Leu Thr Glu Pro Asn Gln 290 295 49PRTInfluenza
A virussource/note="T-cell epitope-peptide F2" 4Gly Ile Leu Gly Phe
Val Phe Thr Leu 1 5 59PRTInfluenza A virussource/note="T-cell
epitope-peptide F2-2" 5Asp Ile Leu Gly Phe Val Phe Thr Leu 1 5
69PRTInfluenza A virussource/note="T-cell epitope-peptide F2-3"
6Asp Ile Leu Gly Phe Asp Phe Thr Leu 1 5 79PRTInfluenza A
virussource/note="T-cell epitope-peptide F2-4" 7Gly Ile Leu Gly Asp
Val Phe Thr Leu 1 5 89PRTInfluenza A virussource/note="T-cell
epitope-peptide F3" 8Ile Leu Arg Gly Ser Val Ala His Lys 1 5
99PRTInfluenza A virussource/note="T-cell epitope-peptide F3-4"
9Ile Leu Arg Phe Ser Val Ala His Lys 1 5 109PRTHuman
cytomegalovirussource/note="T-cell epitope-peptide C2" 10Asn Leu
Val Pro Met Val Ala Thr Val 1 5 11251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 11Lys Glu Phe Ile Leu Arg Phe Ser Val Ala His Lys Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
12251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 12Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ile Leu Gly Phe 35 40 45 Val Phe
Thr Leu Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 13251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 13Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Asn Leu Val Pro Met Val 35 40
45 Ala Thr Val Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 14252PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 14Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Gly Ile Leu
Gly Phe Val Phe Thr Leu Glu Gly Arg 50 55 60 Phe Asn Asn Leu Arg
Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val
Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser
His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp 100 105
110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly
115 120 125 Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp
Leu Met 130 135 140 Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala
Arg Ala Met Leu 145 150 155 160 Arg Phe Val Thr Val Thr Ala Glu Ala
Leu Arg Phe Arg Gln Ile Gln 165 170 175 Arg Gly Phe Arg Thr Thr Leu
Asp Asp Leu Ser Gly Arg Ser Tyr Val 180 185 190 Met Thr Ala Glu Asp
Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 195 200 205 Ser Val Leu
Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 210 215 220 Ile
Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 225 230
235 240 Leu Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
15251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 15Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Ile Leu Gly Phe Val Phe Thr Leu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr
Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala
Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205
Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210
215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile
Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245
250 16251PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 16Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Ile Leu Gly Phe Asp Phe Thr Leu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 17251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 17Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asn Leu Val
Pro Met Val Ala Thr Val Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
18251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 18Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Asn Leu Val Pro Met
Val Ala Thr Val 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 19251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 19Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Gly Ile Leu Gly Asp Val Phe Thr Leu Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Ser His His His Ala Ser
Arg Val Ala Arg 245 250 20260PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 20Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Cys Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Ser His His His Ile Leu Arg Gly Ser Val Ala His Lys Ala
Ser 245 250 255 Arg Val Ala Arg 260 21251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 21Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Asn Leu Val Pro Met 35 40 45 Val Ala Thr Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
22251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 22Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Gly Ile Leu Gly Phe Val Phe Thr Leu Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 23251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 23Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Gly Ile Leu Gly
Phe Val 65 70 75 80 Phe Thr Leu Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 24251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 24Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70
75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe
Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser
Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile
Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr
Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr
Gln Ser Val Ala Arg Gly Ile Leu Gly 145 150 155 160 Phe Val Phe Thr
Leu Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe
Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190
Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195
200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg
Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala
Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val Ala
Arg 245 250 25251PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 25Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Gly Ile Leu Gly Phe Val Phe Thr Leu Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 26251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 26Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Gly Ile Leu Gly 210 215 220 Phe
Val Phe Thr Leu Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
27251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 27Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Gly Ile Leu Gly Phe Val
Phe Thr Leu Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 28251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 28Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Ile Leu Arg Gly Ser Val Ala His Lys Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 29251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 29Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Ile Leu Arg Gly Ser Val 65 70 75 80 Ala His Lys
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
30251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 30Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ile Leu Arg Gly 145 150 155 160 Ser Val Ala
His Lys Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 31251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 31Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Ile Leu Arg Gly Ser Val Ala His Lys Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 32251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 32Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Ile Leu Arg Gly 210 215 220 Ser
Val Ala His Lys Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
33251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 33Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ile Leu Arg Gly Ser Val
Ala His Lys Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 34251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 34Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Val Pro Met Val Ala Thr Val Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 35251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 35Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Val Pro Met Val 65 70 75 80 Ala Thr Val
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
36251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 36Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Asn Leu Val Pro 145 150 155 160 Met Val Ala
Thr Val Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 37251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 37Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val
Thr Gly 65 70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Asn Leu Val Pro Met Val Ala Thr Val Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 38251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 38Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Leu Arg 145 150 155 160 Phe Val Thr Val Thr Ala Glu Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Asn Leu Val Pro 210 215 220 Met
Val Ala Thr Val Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
39251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 39Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Asn Leu Val Pro Met Val
Ala Thr Val Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 40251PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 40Lys Glu Phe Thr Leu
Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val
Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser
Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40
45 Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe
50 55 60 Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Gly Ile
Leu Gly 65 70 75 80 Phe Val Phe Thr Leu Asn Asn Val Phe Tyr Arg Phe
Ala Asp Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val
Thr Leu Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val
Ala Gly Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg 165 170
175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met
180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser
Arg Val Ala Arg 245 250 41251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 41Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Gly Ile 145 150 155
160 Leu Gly Phe Val Phe Thr Leu Ala Leu Arg Phe Arg Gln Ile Gln Arg
165 170 175 Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr
Val Met 180 185 190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly
Arg Leu Ser Ser 195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser
Val Arg Val Gly Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile
Leu Gly Ser Val Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His
Ala Ser Arg Val Ala Arg 245 250 42251PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 42Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr
Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro
Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr Ser Leu Leu Met Ile
Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe Ala Val Asp Val Arg
Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60 Asn Asn Leu Arg Leu
Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65 70 75 80 Phe Val Asn
Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe Ser 85 90 95 His
Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 100 105
110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly Met
115 120 125 Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu
Met Ser 130 135 140 His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg
Ala Met Ile Leu 145 150 155 160 Arg Gly Ser Val Ala His Lys Ala Leu
Arg Phe Arg Gln Ile Gln Arg 165 170 175 Gly Phe Arg Thr Thr Leu Asp
Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185 190 Thr Ala Glu Asp Val
Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser 195 200 205 Val Leu Pro
Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg Ile 210 215 220 Ser
Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile Leu 225 230
235 240 Asn Cys His His His Ala Ser Arg Val Ala Arg 245 250
43251PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 43Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys Thr Tyr Val Asp Ser 1 5 10 15 Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro Leu Gln Thr Ile Ser 20 25 30 Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp Asn 35 40 45 Leu Phe
Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg Phe 50 55 60
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr Gly 65
70 75 80 Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp
Phe Ser 85 90 95 His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu
Ser Gly Asp Ser 100 105 110 Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile Ser Arg Thr Gly Met 115 120 125 Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr Leu Asp Leu Met Ser 130 135 140 His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg Ala Met Leu Arg 145 150 155 160 Phe Asn Leu
Val Pro Met Val Ala Thr Val Phe Arg Gln Ile Gln Arg 165 170 175 Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val Met 180 185
190 Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser Ser
195 200 205 Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly
Arg Ile 210 215 220 Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
Ala Leu Ile Leu 225 230 235 240 Asn Cys His His His Ala Ser Arg Val
Ala Arg 245 250 44193PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 44Gly Ala Asp Asp Val
Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe Ser Ser
Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25 30 Lys
Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp 35 40
45 Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly
50 55 60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly
Gly Val 65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu
Ala Leu Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu
Gly Leu Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val Gly Thr
Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser Arg Val
Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser Val Glu
Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155 160 Val
Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170
175 Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg
180 185 190 Arg 45398PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 45Met Gly Ala Asp Asp
Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe Ser
Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30 Gln
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35 40
45 Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala
50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala
Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val
Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu
Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val Gly
Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser Arg
Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser Val
Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser
Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln 165 170
175 Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val
180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp
Trp Asp 195 200 205 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser
Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser
Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln Tyr
Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu
Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala
Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295
300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly
305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser
Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu
Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe
Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser
Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Lys Thr Gln Pro
Gly Gly Ser His His His His His His 385 390 395 46398PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 46Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe
Val Met Glu 1 5 10 15 Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly
Tyr Val Asp Ser Ile 20 25 30 Gln Lys Gly Ile Leu Gly Phe Val Phe
Thr Leu Gln Gly Asn Tyr Asp 35 40 45 Asp Asp Trp Lys Gly Phe Tyr
Ser Thr Asp Asn Lys Tyr Asp Ala Ala 50 55 60 Gly Tyr Ser Val Asp
Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly 65 70 75 80 Val Val Lys
Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys 85 90 95 Val
Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr 100 105
110 Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe
115 120 125 Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala
Glu Gly 130 135 140 Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln
Ala Lys Ala Leu 145 150 155 160 Ser Val Glu Leu Glu Ile Asn Phe Glu
Thr Arg Gly Lys Arg Gly Gln 165 170 175 Asp Ala Met Tyr Glu Tyr Met
Ala Gln Ala Cys Ala Gly Asn Arg Val 180 185 190 Arg Arg Ser Val Gly
Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 195 200 205 Val Ile Arg
Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 210 215 220 Gly
Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser 225 230
235 240 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala
Leu 245 250 255 Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly
Thr Asn Pro 260 265 270 Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala
Val Asn Val Ala Gln 275 280 285 Val Ile Asp Ser Glu Thr Ala Asp Asn
Leu Glu Lys Thr Thr Ala Ala 290 295 300 Leu Ser Ile Leu Pro Gly Ile
Gly Ser Val Met Gly Ile Ala Asp Gly 305 310 315 320 Ala Val His His
Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 325 330 335 Ser Ser
Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val 340 345 350
Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu 355
360 365 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro
Gly 370 375 380 His Lys Thr Gln Pro Gly Gly Ser His His His His His
His 385 390 395 47398PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 47Met Gly Ala Asp Asp
Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe Ser
Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30 Gln
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35 40
45 Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala
50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala
Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val
Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu
Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val Gly
Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser Arg
Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser Val
Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser
Val Glu Leu Glu Ile Asn Phe Ile Leu Arg Gly Ser Val Ala His 165 170
175 Lys Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val
180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp
Trp Asp 195 200 205 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser
Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser
Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln Tyr
Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu
Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala
Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295
300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly
305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser
Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu
Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe
Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser
Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Lys Thr Gln Pro
Gly Gly Ser His His His His His His 385 390 395 48398PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 48Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe
Val Met Glu 1 5 10 15 Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly
Tyr Val Asp Ser Ile 20 25 30 Gln Lys Gly Ile Gln Lys Pro Lys Ser
Gly Thr Gln Gly Asn Tyr Asp 35 40 45 Asp Asp Trp Lys Gly Phe Tyr
Ser Thr Asp Asn Lys Tyr Asp Ala Ala 50 55 60 Gly Tyr Ser Val Asp
Asn Leu Val Pro Met Val Ala Thr Val Gly Gly 65 70 75 80 Val Val Lys
Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys 85 90 95 Val
Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr 100 105
110 Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe
115 120 125 Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala
Glu Gly 130 135 140 Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln
Ala Lys Ala Leu 145 150 155 160 Ser Val Glu Leu Glu Ile Asn Phe Glu
Thr Arg Gly Lys Arg Gly Gln 165 170 175 Asp Ala Met Tyr Glu Tyr Met
Ala Gln Ala Cys Ala Gly Asn Arg Val 180 185 190 Arg Arg Ser Val Gly
Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp 195 200 205 Val Ile Arg
Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His 210
215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val
Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His
Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu Ser Glu Leu Lys Thr
Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala Gly Ala Asn Tyr Ala
Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val Ile Asp Ser Glu Thr
Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295 300 Leu Ser Ile Leu
Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly 305 310 315 320 Ala
Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu 325 330
335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val
340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile
Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala
Tyr Ser Pro Gly 370 375 380 His Lys Thr Gln Pro Gly Gly Ser His His
His His His His 385 390 395 49512PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 49Met Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val
Lys Pro Gly 1 5 10 15 Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly
Tyr Thr Phe Thr Ser 20 25 30 Tyr Asn Val His Trp Val Lys Gln Thr
Pro Gly Gln Gly Leu Glu Trp 35 40 45 Ile Gly Ala Ile Tyr Pro Gly
Asn Gly Asp Thr Ser Phe Asn Gln Lys 50 55 60 Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val 65 70 75 80 Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95 Cys
Ala Arg Ser Asn Tyr Tyr Gly Ser Ser Tyr Val Trp Phe Phe Asp 100 105
110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser
115 120 125 Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Gln Ile Val
Leu Ser 130 135 140 Gln Ser Pro Thr Ile Leu Ser Ala Ser Pro Gly Glu
Lys Val Thr Met 145 150 155 160 Thr Cys Arg Ala Ser Ser Ser Val Ser
Tyr Met Asp Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Ser Ser Pro Lys
Pro Trp Ile Tyr Ala Thr Ser Asn Leu 180 185 190 Ala Ser Gly Val Pro
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser 195 200 205 Tyr Ser Leu
Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr 210 215 220 Tyr
Cys Gln Gln Trp Ile Ser Asn Pro Pro Thr Phe Gly Ala Gly Thr 225 230
235 240 Lys Leu Glu Leu Lys Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly
Ser 245 250 255 Ser Gly Gly Ala Pro Lys Glu Phe Thr Leu Asp Phe Ser
Thr Ala Lys 260 265 270 Thr Tyr Val Asp Ser Leu Asn Val Ile Arg Ser
Ala Ile Gly Thr Pro 275 280 285 Leu Gln Thr Ile Ser Ser Gly Gly Thr
Ser Leu Leu Met Ile Asp Ser 290 295 300 Asn Leu Val Pro Met Val Ala
Thr Val Asp Val Arg Gly Ile Asp Pro 305 310 315 320 Glu Glu Gly Arg
Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn 325 330 335 Leu Tyr
Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg 340 345 350
Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr 355
360 365 Leu Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly
Ile 370 375 380 Ser Arg Thr Gly Met Gln Ile Asn Arg His Ser Leu Thr
Thr Ser Tyr 385 390 395 400 Leu Asp Leu Met Ser His Ser Gly Thr Ser
Leu Thr Gln Ser Val Ala 405 410 415 Arg Ala Met Leu Arg Phe Val Thr
Val Thr Ala Glu Ala Leu Arg Phe 420 425 430 Arg Gln Ile Gln Arg Gly
Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly 435 440 445 Arg Ser Tyr Val
Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp 450 455 460 Gly Arg
Leu Ser Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val 465 470 475
480 Arg Val Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser
485 490 495 Val Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val
Ala Arg 500 505 510 50511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 50Met Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val
Lys Pro Gly 1 5 10 15 Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser 20 25 30 Tyr Asn Met His Trp Val Lys Gln Thr
Pro Gly Gln Gly Leu Glu Trp 35 40 45 Ile Gly Ala Ile Tyr Pro Gly
Asn Gly Asp Thr Ser Tyr Asn Gln Lys 50 55 60 Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95 Cys
Ala Arg Ala Gln Leu Arg Pro Asn Tyr Trp Tyr Phe Asp Val Trp 100 105
110 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140 Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro Ala
Ile Leu Ser Ala 145 150 155 160 Ser Pro Gly Glu Lys Val Thr Met Thr
Cys Arg Ala Ser Ser Ser Val 165 170 175 Ser Tyr Met His Trp Tyr Gln
Gln Lys Pro Gly Ser Ser Pro Lys Pro 180 185 190 Trp Ile Tyr Ala Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe 195 200 205 Ser Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val 210 215 220 Glu
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ile Ser Asn 225 230
235 240 Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly
Gly 245 250 255 Gly Ser Gly Gly Lys Glu Phe Thr Leu Asp Phe Ser Thr
Ala Lys Thr 260 265 270 Tyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala
Ile Gly Thr Pro Leu 275 280 285 Gln Thr Ile Ser Ser Gly Gly Thr Ser
Leu Leu Met Ile Asp Ser Gly 290 295 300 Ser Gly Asp Asn Leu Phe Ala
Val Asn Leu Val Pro Met Val Ala Thr 305 310 315 320 Val Gly Arg Phe
Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu 325 330 335 Tyr Val
Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe 340 345 350
Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu 355
360 365 Ser Gly Asp Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile
Ser 370 375 380 Arg Thr Gly Met Gln Ile Asn Arg His Ser Leu Thr Thr
Ser Tyr Leu 385 390 395 400 Asp Leu Met Ser His Ser Gly Thr Ser Leu
Thr Gln Ser Val Ala Arg 405 410 415 Ala Met Leu Arg Phe Val Thr Val
Thr Ala Glu Ala Leu Arg Phe Arg 420 425 430 Gln Ile Gln Arg Gly Phe
Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg 435 440 445 Ser Tyr Val Met
Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly 450 455 460 Arg Leu
Ser Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg 465 470 475
480 Val Gly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val
485 490 495 Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val Ala
Arg 500 505 510 51513PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 51Met Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly 1 5 10 15 Ala Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 20 25 30 Tyr
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp 35 40
45 Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys
50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp
Trp Tyr Phe Asn Val Trp 100 105 110 Gly Ala Gly Thr Thr Val Thr Val
Ser Ala Gly Ser Thr Ser Gly Ser 115 120 125 Gly Lys Pro Gly Ser Gly
Glu Gly Ser Thr Lys Gly Gln Ile Val Leu 130 135 140 Ser Gln Ser Pro
Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr 145 150 155 160 Met
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln 165 170
175 Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn
180 185 190 Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser
Gly Thr 195 200 205 Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
Asp Ala Ala Thr 210 215 220 Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro
Pro Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Glu
Phe Pro Lys Pro Ser Thr Pro Pro Gly 245 250 255 Ser Ser Gly Gly Ala
Pro Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 260 265 270 Lys Thr Tyr
Val Asp Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 275 280 285 Pro
Leu Gln Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 290 295
300 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp
305 310 315 320 Pro Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu Ile Val
Glu Arg Asn 325 330 335 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr
Asn Asn Val Phe Tyr 340 345 350 Arg Phe Ala Asp Phe Ser His Val Thr
Phe Pro Gly Thr Asn Leu Val 355 360 365 Pro Met Val Ala Thr Val Ser
Tyr Thr Thr Leu Gln Arg Val Ala Gly 370 375 380 Ile Ser Arg Thr Gly
Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser 385 390 395 400 Tyr Leu
Asp Leu Met Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val 405 410 415
Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 420
425 430 Phe Arg Gln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu
Ser 435 440 445 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu
Thr Leu Asn 450 455 460 Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr
His Gly Gln Asp Ser 465 470 475 480 Val Arg Val Gly Arg Ile Ser Phe
Gly Ser Ile Asn Ala Ile Leu Gly 485 490 495 Ser Val Ala Leu Ile Leu
Asn Cys His His His Ala Ser Arg Val Ala 500 505 510 Arg
52385PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 52Met Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile 20 25 30 Asn Thr Met Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu 35 40 45 Val Ala
Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser Ala His His
Ser Glu Asp Pro Ser Ser 115 120 125 Lys Ala Pro Lys Ala Pro Lys Glu
Phe Thr Leu Asp Phe Ser Thr Ala 130 135 140 Lys Thr Tyr Val Asp Ser
Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 145 150 155 160 Pro Leu Gln
Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 165 170 175 Ser
Asn Leu Val Pro Met Val Ala Thr Val Asp Val Arg Gly Ile Asp 180 185
190 Pro Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn
195 200 205 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val
Phe Tyr 210 215 220 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly
Thr Thr Ala Val 225 230 235 240 Thr Leu Ser Gly Asp Ser Ser Tyr Thr
Thr Leu Gln Arg Val Ala Gly 245 250 255 Ile Ser Arg Thr Gly Met Gln
Ile Asn Arg His Ser Leu Thr Thr Ser 260 265 270 Tyr Leu Asp Leu Met
Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val 275 280 285 Ala Arg Ala
Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg 290 295 300 Phe
Arg Gln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 305 310
315 320 Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr Leu
Asn 325 330 335 Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His Gly
Gln Asp Ser 340 345 350 Val Arg Val Gly Arg Ile Ser Phe Gly Ser Ile
Asn Ala Ile Leu Gly 355 360 365 Ser Val Ala Leu Ile Leu Asn Cys His
His His Ala Ser Arg Val Ala 370 375 380 Arg 385 53387PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 53Met Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr
Tyr Val Asp 1 5 10 15 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr
Pro Leu Gln Thr Ile 20 25 30 Ser Ser Gly Gly Thr Ser Leu Leu Met
Ile Asp Ser Gly Ser Gly Asp 35 40 45 Asn Leu Phe Ala Val Asn Leu
Val Pro Met Val Ala Thr Val Gly Arg 50 55 60 Phe Asn Asn Leu Arg
Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val
Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser
His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp 100 105
110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly
115 120 125 Met Gln Ile Asn Arg His Ser
Leu Thr Thr Ser Tyr Leu Asp Leu Met 130 135 140 Ser His Ser Gly Thr
Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 145 150 155 160 Arg Phe
Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 165 170 175
Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val 180
185 190 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu
Ser 195 200 205 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg
Val Gly Arg 210 215 220 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly
Ser Val Ala Leu Ile 225 230 235 240 Leu Asn Cys His His His Ala Ser
Arg Val Ala Arg Glu Phe Pro Lys 245 250 255 Pro Ser Thr Pro Pro Gly
Ser Ser Gly Gly Ala Pro Met Glu Val Gln 260 265 270 Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg 275 280 285 Leu Ser
Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly 290 295 300
Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile 305
310 315 320 Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly
Arg Phe 325 330 335 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu Gln Met Asn 340 345 350 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Lys Arg Phe Arg 355 360 365 Thr Ala Ala Gln Gly Thr Asp Tyr
Trp Gly Gln Gly Thr Gln Val Thr 370 375 380 Val Ser Ser 385
54390PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 54Met Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile 20 25 30 Asn Thr Met Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu 35 40 45 Val Ala
Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser Glu Phe Pro
Lys Pro Ser Thr Pro Pro 115 120 125 Gly Ser Ser Gly Gly Ala Pro Lys
Glu Phe Thr Leu Asp Phe Ser Thr 130 135 140 Ala Lys Thr Tyr Val Asp
Ser Leu Asn Val Ile Arg Ser Ala Ile Gly 145 150 155 160 Thr Pro Leu
Gln Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile 165 170 175 Asp
Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly Ile 180 185
190 Asp Pro Glu Glu Gly Arg Phe Asn Asn Leu Arg Leu Ile Val Glu Arg
195 200 205 Asn Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn
Val Phe 210 215 220 Tyr Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro
Gly Thr Asn Leu 225 230 235 240 Val Pro Met Val Ala Thr Val Ser Tyr
Thr Thr Leu Gln Arg Val Ala 245 250 255 Gly Ile Ser Arg Thr Gly Met
Gln Ile Asn Arg His Ser Leu Thr Thr 260 265 270 Ser Tyr Leu Asp Leu
Met Ser His Ser Gly Thr Ser Leu Thr Gln Ser 275 280 285 Val Ala Arg
Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu 290 295 300 Arg
Phe Arg Gln Ile Gln Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu 305 310
315 320 Ser Gly Arg Ser Tyr Val Met Thr Ala Glu Asp Val Asp Leu Thr
Leu 325 330 335 Asn Trp Gly Arg Leu Ser Ser Val Leu Pro Asp Tyr His
Gly Gln Asp 340 345 350 Ser Val Arg Val Gly Arg Ile Ser Phe Gly Ser
Ile Asn Ala Ile Leu 355 360 365 Gly Ser Val Ala Leu Ile Leu Asn Cys
His His His Ala Ser Arg Val 370 375 380 Ala Arg Lys Asp Glu Leu 385
390 55652PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 55Met Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly 1 5 10 15 Ala Ser Val
Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser 20 25 30 Tyr
Asn Val His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp 35 40
45 Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Phe Asn Gln Lys
50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Val 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ser Asn Tyr Tyr Gly Ser Ser
Tyr Val Trp Phe Phe Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser Gly Ser Thr Ser 115 120 125 Gly Ser Gly Lys Pro Gly
Ser Gly Glu Gly Ser Gln Ile Val Leu Ser 130 135 140 Gln Ser Pro Thr
Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met 145 150 155 160 Thr
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asp Trp Tyr Gln Gln 165 170
175 Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu
180 185 190 Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly
Thr Ser 195 200 205 Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp
Ala Ala Thr Tyr 210 215 220 Tyr Cys Gln Gln Trp Ile Ser Asn Pro Pro
Thr Phe Gly Ala Gly Thr 225 230 235 240 Lys Leu Glu Leu Lys Glu Phe
Pro Lys Pro Ser Thr Pro Pro Gly Ser 245 250 255 Ser Gly Gly Ala Pro
Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser 260 265 270 Phe Val Met
Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr 275 280 285 Val
Asp Ser Ile Gln Lys Gly Ile Leu Gly Phe Val Phe Thr Leu Gln 290 295
300 Gly Asn Tyr Asp Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys
305 310 315 320 Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro
Leu Ser Gly 325 330 335 Lys Ala Gly Gly Val Val Lys Val Thr Tyr Pro
Gly Leu Thr Lys Val 340 345 350 Leu Ala Leu Lys Val Asp Asn Ala Glu
Thr Ile Lys Lys Glu Leu Gly 355 360 365 Leu Ser Leu Thr Glu Pro Leu
Met Glu Gln Val Gly Thr Glu Glu Phe 370 375 380 Ile Lys Arg Phe Gly
Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro 385 390 395 400 Phe Ala
Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln 405 410 415
Ala Lys Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly 420
425 430 Lys Arg Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys
Ala 435 440 445 Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser
Cys Ile Asn 450 455 460 Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys
Thr Lys Ile Glu Ser 465 470 475 480 Leu Lys Glu His Gly Pro Ile Lys
Asn Lys Met Ser Glu Ser Pro Asn 485 490 495 Lys Thr Val Ser Glu Glu
Lys Ala Lys Gln Tyr Leu Glu Glu Phe His 500 505 510 Gln Thr Ala Leu
Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr 515 520 525 Gly Thr
Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val 530 535 540
Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys 545
550 555 560 Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val
Met Gly 565 570 575 Ile Ala Asp Gly Ala Val His His Asn Thr Glu Glu
Ile Val Ala Gln 580 585 590 Ser Ile Ala Leu Ser Ser Leu Met Val Ala
Gln Ala Ile Pro Leu Val 595 600 605 Gly Glu Leu Val Asp Ile Gly Phe
Ala Ala Tyr Asn Phe Val Glu Ser 610 615 620 Ile Ile Asn Leu Phe Gln
Val Val His Asn Ser Tyr Asn Arg Pro Ala 625 630 635 640 Tyr Ser Pro
Gly His Lys Thr Gln Pro Gly Gly Ser 645 650 56651PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 56Met Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Val
Lys Pro Gly 1 5 10 15 Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser 20 25 30 Tyr Asn Met His Trp Val Lys Gln Thr
Pro Gly Gln Gly Leu Glu Trp 35 40 45 Ile Gly Ala Ile Tyr Pro Gly
Asn Gly Asp Thr Ser Tyr Asn Gln Lys 50 55 60 Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95 Cys
Ala Arg Ala Gln Leu Arg Pro Asn Tyr Trp Tyr Phe Asp Val Trp 100 105
110 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140 Gly Gly Ser Asp Ile Val Leu Ser Gln Ser Pro Ala
Ile Leu Ser Ala 145 150 155 160 Ser Pro Gly Glu Lys Val Thr Met Thr
Cys Arg Ala Ser Ser Ser Val 165 170 175 Ser Tyr Met His Trp Tyr Gln
Gln Lys Pro Gly Ser Ser Pro Lys Pro 180 185 190 Trp Ile Tyr Ala Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe 195 200 205 Ser Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val 210 215 220 Glu
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ile Ser Asn 225 230
235 240 Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly
Gly 245 250 255 Gly Ser Gly Gly Gly Ala Asp Asp Val Val Asp Ser Ser
Lys Ser Phe 260 265 270 Val Met Glu Asn Phe Ser Ser Tyr His Gly Thr
Lys Pro Gly Tyr Val 275 280 285 Asp Ser Ile Gln Lys Gly Ile Gln Lys
Pro Lys Ser Gly Thr Gln Gly 290 295 300 Asn Tyr Asp Asp Asp Trp Lys
Gly Phe Tyr Ser Thr Asp Asn Lys Tyr 305 310 315 320 Asp Ala Ala Gly
Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys 325 330 335 Ala Gly
Gly Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu 340 345 350
Ala Leu Lys Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu 355
360 365 Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe
Ile 370 375 380 Lys Arg Phe Gly Asp Gly Ala Ser Arg Val Val Leu Ser
Leu Pro Phe 385 390 395 400 Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile
Asn Asn Trp Glu Gln Ala 405 410 415 Lys Ala Leu Ser Val Glu Leu Glu
Ile Asn Phe Ile Leu Arg Gly Ser 420 425 430 Val Ala His Lys Ala Met
Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly 435 440 445 Asn Arg Val Arg
Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu 450 455 460 Asp Trp
Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu 465 470 475
480 Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys
485 490 495 Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe
His Gln 500 505 510 Thr Ala Leu Glu His Pro Glu Leu Ser Glu Leu Lys
Thr Val Thr Gly 515 520 525 Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr
Ala Ala Trp Ala Val Asn 530 535 540 Val Ala Gln Val Ile Asp Ser Glu
Thr Ala Asp Asn Leu Glu Lys Thr 545 550 555 560 Thr Ala Ala Leu Ser
Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile 565 570 575 Ala Asp Gly
Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser 580 585 590 Ile
Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly 595 600
605 Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile
610 615 620 Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro
Ala Tyr 625 630 635 640 Ser Pro Gly His Lys Thr Gln Pro Gly Gly Ser
645 650 57653PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 57Met Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly 1 5 10 15 Ala Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 20 25 30 Tyr
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp 35 40
45 Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys
50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp
Trp Tyr Phe Asn Val Trp 100 105 110 Gly Ala Gly Thr Thr Val Thr Val
Ser Ala Gly Ser Thr Ser Gly Ser 115 120 125 Gly Lys Pro Gly Ser Gly
Glu Gly Ser Thr Lys Gly Gln Ile Val Leu 130 135 140 Ser Gln Ser Pro
Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr 145 150 155 160 Met
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe Gln 165 170
175 Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn
180 185 190 Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser
Gly Thr 195 200 205 Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
Asp Ala Ala Thr 210 215 220 Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro
Pro Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Glu
Phe Pro Lys Pro Ser Thr Pro Pro Gly 245 250 255 Ser Ser Gly Gly Ala
Pro Gly Ala Asp Asp Val Val Asp Ser Ser Lys 260 265 270 Ser Phe Val
Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly 275 280 285 Tyr
Val Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr 290
295
300 Gln Gly Asn Tyr Asp Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn
305 310 315 320 Lys Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Leu Val
Pro Met Val 325 330 335 Ala Thr Val Gly Gly Val Val Lys Val Thr Tyr
Pro Gly Leu Thr Lys 340 345 350 Val Leu Ala Leu Lys Val Asp Asn Ala
Glu Thr Ile Lys Lys Glu Leu 355 360 365 Gly Leu Ser Leu Thr Glu Pro
Leu Met Glu Gln Val Gly Thr Glu Glu 370 375 380 Phe Ile Lys Arg Phe
Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu 385 390 395 400 Pro Phe
Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu 405 410 415
Gln Ala Lys Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg 420
425 430 Gly Lys Arg Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala
Cys 435 440 445 Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu
Ser Cys Ile 450 455 460 Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr
Lys Thr Lys Ile Glu 465 470 475 480 Ser Leu Lys Glu His Gly Pro Ile
Lys Asn Lys Met Ser Glu Ser Pro 485 490 495 Asn Lys Thr Val Ser Glu
Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe 500 505 510 His Gln Thr Ala
Leu Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val 515 520 525 Thr Gly
Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala 530 535 540
Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu 545
550 555 560 Lys Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser
Val Met 565 570 575 Gly Ile Ala Asp Gly Ala Val His His Asn Thr Glu
Glu Ile Val Ala 580 585 590 Gln Ser Ile Ala Leu Ser Ser Leu Met Val
Ala Gln Ala Ile Pro Leu 595 600 605 Val Gly Glu Leu Val Asp Ile Gly
Phe Ala Ala Tyr Asn Phe Val Glu 610 615 620 Ser Ile Ile Asn Leu Phe
Gln Val Val His Asn Ser Tyr Asn Arg Pro 625 630 635 640 Ala Tyr Ser
Pro Gly His Lys Thr Gln Pro Gly Gly Ser 645 650 58525PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 58Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Ala Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Ile Thr Phe Ser Ile 20 25 30 Asn Thr Met Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Gln Arg Glu Leu 35 40 45 Val Ala Leu Ile Ser Ser Ile
Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys
Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Gln Val Thr Val Ser Ser Ala His His Ser Glu Asp Pro Ser Ser
115 120 125 Lys Ala Pro Lys Ala Pro Gly Ala Asp Asp Val Val Asp Ser
Ser Lys 130 135 140 Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His Gly
Thr Lys Pro Gly 145 150 155 160 Tyr Val Asp Ser Ile Gln Lys Gly Ile
Leu Gly Phe Val Phe Thr Leu 165 170 175 Gln Gly Asn Tyr Asp Asp Asp
Trp Lys Gly Phe Tyr Ser Thr Asp Asn 180 185 190 Lys Tyr Asp Ala Ala
Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser 195 200 205 Gly Lys Ala
Gly Gly Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys 210 215 220 Val
Leu Ala Leu Lys Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu 225 230
235 240 Gly Leu Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly Thr Glu
Glu 245 250 255 Phe Ile Lys Arg Phe Gly Asp Gly Ala Ser Arg Val Val
Leu Ser Leu 260 265 270 Pro Phe Ala Glu Gly Ser Ser Ser Val Glu Tyr
Ile Asn Asn Trp Glu 275 280 285 Gln Ala Lys Ala Leu Ser Val Glu Leu
Glu Ile Asn Phe Glu Thr Arg 290 295 300 Gly Lys Arg Gly Gln Asp Ala
Met Tyr Glu Tyr Met Ala Gln Ala Cys 305 310 315 320 Ala Gly Asn Arg
Val Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile 325 330 335 Asn Leu
Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu 340 345 350
Ser Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro 355
360 365 Asn Lys Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu
Phe 370 375 380 His Gln Thr Ala Leu Glu His Pro Glu Leu Ser Glu Leu
Lys Thr Val 385 390 395 400 Thr Gly Thr Asn Pro Val Phe Ala Gly Ala
Asn Tyr Ala Ala Trp Ala 405 410 415 Val Asn Val Ala Gln Val Ile Asp
Ser Glu Thr Ala Asp Asn Leu Glu 420 425 430 Lys Thr Thr Ala Ala Leu
Ser Ile Leu Pro Gly Ile Gly Ser Val Met 435 440 445 Gly Ile Ala Asp
Gly Ala Val His His Asn Thr Glu Glu Ile Val Ala 450 455 460 Gln Ser
Ile Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu 465 470 475
480 Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu
485 490 495 Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn
Arg Pro 500 505 510 Ala Tyr Ser Pro Gly His Lys Thr Gln Pro Gly Gly
Ser 515 520 525 59527PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 59Met Gly Ala Asp Asp
Val Val Asp Ser Ser Lys Ser Phe Val Met Glu 1 5 10 15 Asn Phe Ser
Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile 20 25 30 Gln
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp 35 40
45 Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala
50 55 60 Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala
Gly Gly 65 70 75 80 Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val
Leu Ala Leu Lys 85 90 95 Val Asp Asn Ala Glu Thr Ile Lys Lys Glu
Leu Gly Leu Ser Leu Thr 100 105 110 Glu Pro Leu Met Glu Gln Val Gly
Thr Glu Glu Phe Ile Lys Arg Phe 115 120 125 Gly Asp Gly Ala Ser Arg
Val Val Leu Ser Leu Pro Phe Ala Glu Gly 130 135 140 Ser Ser Ser Val
Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu 145 150 155 160 Ser
Val Glu Leu Glu Ile Asn Phe Ile Leu Arg Gly Ser Val Ala His 165 170
175 Lys Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val
180 185 190 Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp
Trp Asp 195 200 205 Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser
Leu Lys Glu His 210 215 220 Gly Pro Ile Lys Asn Lys Met Ser Glu Ser
Pro Asn Lys Thr Val Ser 225 230 235 240 Glu Glu Lys Ala Lys Gln Tyr
Leu Glu Glu Phe His Gln Thr Ala Leu 245 250 255 Glu His Pro Glu Leu
Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro 260 265 270 Val Phe Ala
Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln 275 280 285 Val
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala 290 295
300 Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly
305 310 315 320 Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser
Ile Ala Leu 325 330 335 Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu
Val Gly Glu Leu Val 340 345 350 Asp Ile Gly Phe Ala Ala Tyr Asn Phe
Val Glu Ser Ile Ile Asn Leu 355 360 365 Phe Gln Val Val His Asn Ser
Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 370 375 380 His Lys Thr Gln Pro
Gly Gly Ser Glu Phe Pro Lys Pro Ser Thr Pro 385 390 395 400 Pro Gly
Ser Ser Gly Gly Ala Pro Met Glu Val Gln Leu Val Glu Ser 405 410 415
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala 420
425 430 Ala Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr Arg
Gln 435 440 445 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser
Ser Ile Gly 450 455 460 Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg 465 470 475 480 Asp Asn Ala Lys Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro 485 490 495 Glu Asp Thr Ala Val Tyr
Tyr Cys Lys Arg Phe Arg Thr Ala Ala Gln 500 505 510 Gly Thr Asp Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 515 520 525
60530PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 60Met Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile 20 25 30 Asn Thr Met Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu 35 40 45 Val Ala
Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Lys Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser Glu Phe Pro
Lys Pro Ser Thr Pro Pro 115 120 125 Gly Ser Ser Gly Gly Ala Pro Gly
Ala Asp Asp Val Val Asp Ser Ser 130 135 140 Lys Ser Phe Val Met Glu
Asn Phe Ser Ser Tyr His Gly Thr Lys Pro 145 150 155 160 Gly Tyr Val
Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly 165 170 175 Thr
Gln Gly Asn Tyr Asp Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp 180 185
190 Asn Lys Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Leu Val Pro Met
195 200 205 Val Ala Thr Val Gly Gly Val Val Lys Val Thr Tyr Pro Gly
Leu Thr 210 215 220 Lys Val Leu Ala Leu Lys Val Asp Asn Ala Glu Thr
Ile Lys Lys Glu 225 230 235 240 Leu Gly Leu Ser Leu Thr Glu Pro Leu
Met Glu Gln Val Gly Thr Glu 245 250 255 Glu Phe Ile Lys Arg Phe Gly
Asp Gly Ala Ser Arg Val Val Leu Ser 260 265 270 Leu Pro Phe Ala Glu
Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp 275 280 285 Glu Gln Ala
Lys Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr 290 295 300 Arg
Gly Lys Arg Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala 305 310
315 320 Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser
Cys 325 330 335 Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys
Thr Lys Ile 340 345 350 Glu Ser Leu Lys Glu His Gly Pro Ile Lys Asn
Lys Met Ser Glu Ser 355 360 365 Pro Asn Lys Thr Val Ser Glu Glu Lys
Ala Lys Gln Tyr Leu Glu Glu 370 375 380 Phe His Gln Thr Ala Leu Glu
His Pro Glu Leu Ser Glu Leu Lys Thr 385 390 395 400 Val Thr Gly Thr
Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp 405 410 415 Ala Val
Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu 420 425 430
Glu Lys Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val 435
440 445 Met Gly Ile Ala Asp Gly Ala Val His His Asn Thr Glu Glu Ile
Val 450 455 460 Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln
Ala Ile Pro 465 470 475 480 Leu Val Gly Glu Leu Val Asp Ile Gly Phe
Ala Ala Tyr Asn Phe Val 485 490 495 Glu Ser Ile Ile Asn Leu Phe Gln
Val Val His Asn Ser Tyr Asn Arg 500 505 510 Pro Ala Tyr Ser Pro Gly
His Lys Thr Gln Pro Gly Gly Ser Lys Asp 515 520 525 Glu Leu 530
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