U.S. patent application number 15/675493 was filed with the patent office on 2018-08-09 for bispecific scfv immunofusion (bif).
The applicant listed for this patent is SCOTT & WHITE HEALTHCARE (SWH). Invention is credited to Shu-Ru Kuo, Jen-Sing Liu.
Application Number | 20180222996 15/675493 |
Document ID | / |
Family ID | 49584484 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180222996 |
Kind Code |
A1 |
Liu; Jen-Sing ; et
al. |
August 9, 2018 |
BISPECIFIC SCFV IMMUNOFUSION (BIF)
Abstract
Certain embodiments are directed to a bispecific immunoglobulin
that is capable of binding cell surface molecules on a target cell,
such as cancer cells, and cell surface molecules on immune effector
cell, such as cytotoxic T lymphocytes, resulting in the targeted
killing of target cells.
Inventors: |
Liu; Jen-Sing; (Austin,
TX) ; Kuo; Shu-Ru; (Temple, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SCOTT & WHITE HEALTHCARE (SWH) |
Temple |
TX |
US |
|
|
Family ID: |
49584484 |
Appl. No.: |
15/675493 |
Filed: |
August 11, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14402009 |
Nov 18, 2014 |
9745381 |
|
|
PCT/US2013/041739 |
May 18, 2013 |
|
|
|
15675493 |
|
|
|
|
61649211 |
May 18, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/64 20130101;
C07K 2317/52 20130101; C07K 2317/622 20130101; C07K 16/2809
20130101; C07K 2317/56 20130101; C07K 2317/31 20130101; C07K
16/2896 20130101; C07K 16/30 20130101; C07K 16/2866 20130101; C07K
2317/732 20130101; A61P 35/02 20180101; C07K 2317/92 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28 |
Claims
1. A polypeptide comprising a first target binding domain that
specifically binds a cancer cell, a second effector binding domain
that specifically binds an immunologic effector, and an
immunoglobulin constant region linker operatively coupling the
first and second binding domain.
2. The polypeptide of claim 1, wherein the target binding domain
binds a colorectal, lung, breast, kidney, brain, prostate,
pancreas, or blood cancer cell or solid malignant tumor cell.
3. The polypeptide of claim 2, wherein the target binding domain
binds a leukemia cell.
4. The polypeptide of claim 3, wherein the target binding domain
specifically binds cells expressing CD123, prostate specific
membrane antigen (PSMA), TEM8, or CD33.
5. The polypeptide of claim 4, wherein the target binding domain
specifically binds CD123.
6. The polypeptide of claim 5, wherein the target binding domain
comprises variable regions that are 95% identical to variable
regions of SEQ ID NO:2.
7. The polypeptide of claim 5, wherein the target binding domain
comprises variable regions having an amino acid sequence of the
variable regions of SEQ ID NO:2.
8. The polypeptide of claim 1, wherein the target binding domain
has the amino sequence of SEQ ID NO:2.
9. The polypeptide of claim 1, wherein the immune effector is a
lymphocyte.
10. The polypeptide of claim 9, wherein the lymphocyte is a CD3+
cytotoxic T lymphocyte.
11. The polypeptide of claim 1, wherein the effector binding domain
specifically binds CD3.
12. The polypeptide of claim 11, wherein the effector binding
domain comprises hypervariable regions that are 95% identical to
the hypervariable regions of SEQ ID NO:3.
13. The polypeptide of claim 11, wherein the effector binding
domain comprises hypervariable regions having an amino acid
sequence identical to SEQ ID NO:3.
14. The polypeptide of claim 1, wherein the immunoglobulin constant
region is a human IgG constant region.
15. The polypeptide of claim 14, wherein the human IgG constant
region is a hinge-C.sub.H2-C.sub.H3 segment of human IgG1.
16. The polypeptide of claim 14, wherein the immunoglobulin
constant region has an amino acid sequence that is 95% identical to
SEQ ID NO:4.
17. The polypeptide of claim 14, wherein the immunoglobulin
constant region has an amino acid sequence identical to SEQ ID
NO:4.
18. A polypeptide having an amino acid sequence that is 90%
identical to SEQ ID NO:1.
19. A method for treating cancer comprising providing an effective
amount of the polypeptide of claim 1 to a cancer patient.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/402,009, filed Nov. 18, 2014, which is a
U.S. national stage filing of International Application Serial
Number PCT/US2013/041739, filed May 18, 2013, which claims priority
to U.S. Provisional Patent Application Ser. No. 61/649,211, filed
May 18, 2012. Priority to each of these applications is claimed and
each application is incorporated herein by reference in its
entirety.
[0002] First generation bispecific antibodies were developed over
20 years ago. Since then a number of clinical studies have tested
bispecific antibodies engineered to target cancer cell surface
antigens. This group of anti-cancer fusion proteins contains two or
more functional domains that localize immunological effector cells
in the proximity of targeted cancer cells to achieve anti-cancer
activity.
[0003] One therapeutic agent of this family, Catumaxomab, was
approved in 2009 by the European Union for the treatment of
malignant ascites. Catumaxomab is a hybrid immunoglobulin of mouse
and rat antibodies having a first binding site for EpCAM on tumor
cell surface and a second binding site for CD3 of T lymphocytes.
While the activated T cells provide the major cellular
cytotoxicity, the Fc region through Fc receptors also bring
macrophage, nature killer (NK) cells, and granulocytes to targeted
cells activating the antibody-dependent cellular cytotoxicity
(ADCC) responses. However, therapeutic antibodies carrying Fc of
animal origins are typically immunogenic in humans and treated
patients could adversely react to these therapeutic agents,
reducing their half-life and efficacies. Murine antibodies are also
associated with the generation of severe allergic reactions.
[0004] As bispecific antibody technology developed, a different
group of fusion proteins named bispecific T-cell engagers (BiTE)
were generated by connecting two antibody single chain variable
regions (scFv) only (no Fc amino acid segments were included) with
a flexible linker, one scFv binds targeted cells and the other
binds CD3 on T cell surface. One BiTE, blinatumomab, with CD19xCD3
bi-specific binding activities showed promising results in Phase II
clinical trials for patients with minimal residual disease in
B-lineage acute lymphoblastic leukemia.
[0005] In spite of these advancements in bispecific antibody
technology, there remains a need for additional cancer
therapeutics, particularly those that efficiently target and kill
cancer cells either directly or indirectly.
SUMMARY
[0006] Certain embodiments are directed to bispecific
immunoglobulin therapeutics (bispecific scFV immunofusions, BIf)
and methods of using such therapeutics. In certain aspects a BIf is
a polypeptide comprising a first target binding domain that
specifically binds a cancer cell, a second effector binding domain
that specifically binds an immunologic effector, and an
immunoglobulin constant region linker operatively coupling the
first and second binding domain. As used herein the terms "specific
binding" or "specifically binds" refers to a polypeptide domain
that has a binding affinity predominately for a target cell type or
protein and binds its target with a 10, 100, 1000 fold or greater
affinity as compared to non-target cells or proteins.
[0007] In certain aspects, a target binding domain specifically
binds to a cell surface polypeptide that is specifically expressed
by a cancer cell or is over expressed by a cancer cell. In a
further aspect, the cancer cell is a leukemia cell, including but
not limited to acute myelogenous leukemia (AML), chronic
myelogenous leukemia (CML), or plasmacytoid leukemia. In a further
aspect the cancer cell can be, but is not limited to a colorectal,
lung, breast, kidney, brain, prostate, pancreas, or blood cancer
cell or a solid malignant tumor cell.
[0008] In certain aspects, the cell surface polypeptide is CD123.
CD123 is also known as the interleukin-3 receptor (IL-3). IL-3 is a
soluble cytokine that can be secreted by activated T cells. One
example of CD123 binding domain comprises the variable regions of
the anti-CD123 antibody 12F1 (amino acids 1 to 113, and 132 to 250
of SEQ ID NO:2). In certain aspects, the CD123 binding domain
variable regions are, independently 80, 85, 90, 95, 98, or 100%
identical to the variable regions of SEQ ID NO:2. In certain
aspects, the CD123 binding domain is 80, 85, 90, 95, 98, or 100%
identical to SEQ ID NO:2.
[0009] In further aspects, the cell surface polypeptide is tumor
endothelial marker 8 ("TEM8") polypeptide. TEM8 is an 85 kDa
integrin-like cell surface receptor that was originally identified
as one of several unrelated genes (called TEM1-TEM9) overexpressed
in vascular endothelial cells derived from tumor versus normal
colorectal tissues. TEM8 is overexpressed in the blood vessels of a
variety of human cancer types. One example of a TEM8 binding domain
comprises the variable light chain region
DIVMTQTPPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSN
LASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRA (SEQ ID
NO:14) and/or the variable heavy chain region
QVKLEESGAELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVISTYFG
DATYNQKFKGKATMTVDSSSTAYMELARLTSEDSAIYYCAREDYVPFAYWGQGT LVTVSA (SEQ
ID NO:15).
[0010] In still further aspects, the cell surface polypeptide is
prostate-specific membrane antigen ("PSMA") polypeptide. PSMA (also
known as Glutamate carboxypeptidase II) is a type 2 integral
membrane glycoprotein found in prostate tissues and a few other
tissues. One example of a PSMA binding domain comprises the
variable light chain region
WDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRH
TGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK (SEQ ID NO:16)
and/or the variable heavy chain region
EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGT
TYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTV SS (SEQ ID
NO:17).
[0011] In other embodiments, the cell surface polypeptide is a CD33
polypeptide. CD33 is a 67 kDa cell-surface antigen specifically
expressed on myeloid cells including myeloid leukemia cells. It is
the smallest member of the siglec (sialic acid-binding Ig-related
lectins) family. One example of a CD33 binding comprises variable
light chain region
DIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLESGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSSITFGQGTKLEIKR (SEQ ID NO:18)
and/or variable chain region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISW
VRQAPGQGLEWMGRIIPILGVANYAQKFQGRVTITADKSTRTAYMELSSLRSEDTAV
YYCARNWADAFDIWGEGTMVTVSS (SEQ ID NO:19).
[0012] Certain aspects can include target binding domains that
specifically bind cancer antigens that include, but are not limited
to, MAGE (including but not limited to MAGE3, MAGEA6, MAGEA10),
NY-ESO-1, gp100, tyrosinase, EGFR, PSA, VEG-F, PDGFR, KIT, CEA,
HER2/neu, Muc-1, hTERT, MART1, TRP-1, and TRP-2.
[0013] In certain aspects, a BIf comprises a second
effector-binding domain that binds an immunologic effector cell. In
certain aspects the immunologic effector cell is a cytotoxic T
lymphocyte. In certain aspects, the effector binding domain binds
the CD3 protein. CD3 is a T-cell co-receptor that is composed of
four distinct polypeptide chains. In mammals, the complex contains
a CD3.gamma. chain, a CD3.delta. chain, and two CD3.epsilon.
chains. These chains associate with a molecule known as the T-cell
receptor (TCR) and the .zeta.-chain to generate an activation
signal in T-cells. In a further aspect, the effector binding domain
comprises the variable regions of UCHT1 (amino acids 1 to 107 and
126 to 247 of SEQ ID NO:3). In certain aspects, the CD3 binding
domain variable regions are independently 80, 85, 90, 95, 98, or
100% identical to the variable regions of SEQ ID NO:3. In certain
aspects, the CD3 binding domain is 80, 85, 90, 95, 98, or 100%
identical to SEQ ID NO:3.
[0014] In certain aspects, the variable regions of a binding domain
are operatively linked by a peptide linker. In certain aspects, the
target binding domain and the effector binding domain are
operatively linked by a linker. The linker can be designed for
optimal mammalian cell expression. In certain aspects the binding
domains are linked using a hinge-C.sub.H2-C.sub.H3 domain or a
hinge-C.sub.H3 domain of human IgG1 constant region as a linker. In
certain aspects, the IgG1 constant region is human. In certain
aspects, the immunoglobulin constant region is 80, 85, 90, 95, 98,
or 100% identical to SEQ ID NO:4.
[0015] Certain embodiments are directed to an immunofusion
comprising a target binding domain operably linked by a
hinge-C.sub.H2-C.sub.H3 domain or a hinge-C.sub.H3 domain of an
immunoglobulin constant region to an effector binding domain that
specifically binds a CD3 expressing lymphocyte.
[0016] The term "operatively linked" or "operably linked" or the
like, when used to describe fusion proteins, refer to polypeptide
sequences that are placed in a physical and functional relationship
to each other. In certain embodiments, the functions of the
polypeptide components of the fusion molecule are unchanged
compared to the functional activities of the parts in
isolation.
[0017] In certain embodiments the first binding domain, linker, and
second binding domain are comprised as a fusion protein. In certain
aspects, the BIf is 80, 85, 90, 95, 98, or 100% identical to SEQ ID
NO:1.
[0018] In certain aspects the nucleic acids encoding the binding
domains and/or linker are codon optimized. In further aspects, the
amino acid sequence of the binding domains and/or linker is human
or humanized.
[0019] In certain embodiments, a cancer patient is treated for
cancer by providing an effective amount of a BIf described herein.
The term "providing" is used according to its ordinary meaning to
indicate "to supply or furnish for use." In some embodiments, the
protein is provided directly by administering the protein, while in
other embodiments, the protein is effectively provided by
administering a nucleic acid that encodes the protein.
[0020] The term "antibody" or "immunoglobulin" is used to include
intact antibodies and binding fragments/segments thereof.
Typically, fragments compete with the intact antibody from which
they were derived for specific binding to an antigen. Fragments
include separate heavy chains, light chains, Fab, Fab' F(ab')2,
Fabc, and Fv. In certain embodiments the polypeptides are single
chain Fv (scFV). In a further aspect the scFV can be further
modified by a carboxy terminal fusion with a second antibody
binding domain. Fragments/segments are produced by recombinant DNA
techniques, or by enzymatic or chemical separation of intact
immunoglobulins. The term "antibody" also includes one or more
immunoglobulin chains that are chemically conjugated to, or
expressed as, fusion proteins. The term "antibody" also includes a
bispecific antibody. A bispecific or bifunctional antibody is an
artificial hybrid antibody having two different heavy/light chain
pairs and two different binding sites.
[0021] The term "humanized antibody" refers to antibodies in which
the framework and/or "complementarity determining regions" (CDR)
are presented on an immunoglobulin of a different species as
compared to that of the parent immunoglobulin from which the CDR
was derived. In a preferred embodiment, a murine CDR is grafted
into the framework region of a human antibody to prepare the
"humanized antibody." See, e.g., Riechmann, L., et al., Nature 332
(1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985)
268-270. Other forms of "humanized antibodies" encompassed by the
present invention are those in which the constant region has been
additionally modified or changed from that of the original antibody
to generate the properties according to the invention, especially
in regard to C1q binding and/or Fc receptor (FcR) binding.
[0022] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germ line immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human
antibodies can also be produced in transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
or a selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)
2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;
Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human
antibodies can also be produced in phage display libraries
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597).
The techniques of Cole, et al. and Boerner, et al., are also
available for the preparation of human monoclonal antibodies (Cole,
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).
The term "human antibody" as used herein also comprises such
antibodies that are modified in the constant region to generate the
properties according to the invention, especially in regard to C1q
binding and/or FcR binding, e.g. by "class switching" i.e. change
or mutation of Fc parts (e.g. from IgG1 to IgG4 and/or IgG1/IgG4
mutation).
[0023] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NS0 or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions in a rearranged
form. The recombinant human antibodies according to the invention
have been subjected to in vivo somatic hypermutation. Thus, the
amino acid sequences of the VH and VL regions of the recombinant
antibodies are sequences that, while derived from and related to
human germ line VH and VL sequences, may not naturally exist within
the human antibody germ line repertoire in vivo.
[0024] The "variable domain" (including the variable domain of a
light chain (VL) and variable region of a heavy chain (VH)) as used
herein denotes each of the pair of light and heavy chains that are
involved directly in binding the antibody to the antigen. The
domains of variable human light and heavy chains have the same
general structure and each domain comprises four framework (FR)
regions whose sequences are widely conserved and connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.-sheet conformation and
the CDRs may form loops connecting the .beta.-sheet structure. The
CDRs in each chain are held in their three-dimensional structure by
the framework regions and form together with the CDRs from the
other chain the antigen binding site. The antibody heavy and light
chain CDR3 regions play a particularly important role in the
binding specificity/affinity of the antibodies according to the
invention and therefore provide a further object of the
invention.
[0025] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDRs on each chain are separated by such framework amino acids.
Especially, CDR3 of the heavy chain is the region which contributes
most to antigen binding. CDR and FR regions are determined
according to the standard definition of Kabat et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991).
[0026] The term "constant region" as used within the current
applications denotes the sum of the domains of an antibody other
than the variable region. The constant region is not involved
directly in binding of an antigen, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies are divided in the
classes: IgA, IgD, IgE, IgG and IgM, and several of these may be
further divided into subclasses, such as IgG1, IgG2, IgG3, and
IgG4, IgA1 and IgA2. The heavy chain constant regions that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
light chain constant regions (CL) which can be found in all five
antibody classes are called .kappa. (kappa) and .lamda.
(lambda).
[0027] The term "constant region derived from human origin" as used
in the current application denotes a constant heavy chain region of
a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a
constant light chain kappa or lambda region. Such constant regions
are well known in the state of the art and e.g. described by Kabat,
E. A., (see e.g. Johnson, G. and Wu, T. T., Nucleic Acids Res. 28
(2000) 214-218; Kabat, E. A., et al., Proc. Natl. Acad. Sci. USA 72
(1975) 2785-2788).
[0028] The phrase that a molecule "specifically binds" to a target
refers to a binding reaction that is determinative of the presence
of the molecule in the presence of a heterogeneous population of
other biologics. Thus, under designated conditions, a specified
molecule binds preferentially to a particular target and does not
bind in a significant amount to other biologics present in the
sample. Specific binding of an antibody to a target under such
conditions requires the antibody be selected for its specificity to
the target. A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein. See, e.g., Harlow and Lane (1988), for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity. Specific binding between two entities
means an affinity of at least 10.sup.-6, 10.sup.-7, 10.sup.-8,
10.sup.-9, 10.sup.-10 M , or greater.
[0029] The term "isolated" can refer to a nucleic acid or
polypeptide that is substantially free of cellular material,
bacterial material, viral material, or culture medium (when
produced by recombinant DNA techniques) of their source of origin,
or chemical precursors or other chemicals (when chemically
synthesized). Moreover, an isolated compound refers to one that can
be administered to a subject as an isolated compound; in other
words, the compound may not simply be considered "isolated" if it
is adhered to a column or embedded in an agarose gel. Moreover, an
"isolated nucleic acid fragment" or "isolated peptide" is a nucleic
acid or protein fragment that is not naturally occurring as a
fragment and/or is not typically in the functional state.
[0030] Moieties of the invention, such as polypeptides, may be
conjugated or linked covalently or noncovalently to other moieties
such as adjuvants, proteins, peptides, supports, fluorescence
moieties, or labels. The term "conjugate" or "immunoconjugate" is
broadly used to define the operative association of one moiety with
another agent and is not intended to refer solely to any type of
operative association, and is particularly not limited to chemical
"conjugation." Recombinant fusion proteins are particularly
contemplated.
[0031] Other embodiments of the invention are discussed throughout
this application. Any embodiment discussed with respect to one
aspect of the invention applies to other aspects of the invention
as well and vice versa. Each embodiment described herein is
understood to be embodiments of the invention that are applicable
to all aspects of the invention. It is contemplated that any
embodiment discussed herein can be implemented with respect to any
method or composition of the invention, and vice versa.
Furthermore, compositions and kits of the invention can be used to
achieve methods of the invention.
[0032] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0033] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0034] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0035] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0036] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0037] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of the specification
embodiments presented herein.
[0038] FIG. 1A-1C. 12F1scFv-Fc and CD123xCD3 BIf. Schematic diagram
of 12F1scFv-Fc and CD123xCD3 BIf are shown in (A) and (B),
respectively. (C) SDS-PAGE (12.5%) of purified 12F1scFv-Fc and
CD123xCD3 BIf under reduced (right panel) and non-reduced (left
panel) conditions.
[0039] FIG. 2A-2D. Cell surface binding of 12F1scFv-Fc and
CD123xCD3 BIf. Binding of 1 .mu.g/ml of (A) 12F1; (B) CD123xCD3 BIf
and (C) 12F1scFv-Fc to CHO-CD123 was analyzed by flow cytometry
with PE-conjugated secondary antibodies. The dotted histogram
represented the reaction with human IgG1 control. The y-axis
represented cell counts. (D) 96-well plate coated with
5.times.10.sup.4 CHO-CD123 cells/well was fixed and probed by
serial titrations of 12F1 (square); 12F1scFv-Fc (triangle); and
CD123xCD3 BIf (circle) and detected with HRP-conjugated
goat-anti-mouse (12F1) or goat-anti-human (12F1scFv-Fc and
CD123xCD3 BIf) antibodies. The average of three sets of reactions
was plotted by GraphicPad Prism.
[0040] FIG. 3A-3C. T-cell surface binding of CD123xCD3 BIf. Binding
of 1 .mu.g/ml of (A) CD123xCD3 BIf; and (B) UCHT1 to Jurkat T cells
was analyzed by flow cytometry with PE-conjugated secondary
antibodies. The dotted histogram represented the reaction with
human IgG1 control. The y-axis represented cell counts. (C) Serials
titrations of UCHT1 (square) and CD123xCD3 BIf (triangle) were used
in flow cytometric analysis with Jurkat T cells. The mean X-axis
values were plotted versus protein concentrations for Kd
analysis.
[0041] FIG. 4A-4B. Fluorescent images of CellTrace-labeled cells.
4.times.10.sup.4 CFSE-labeled CHO-K1 or CHO-CD123 cells were seeded
in each well of a 96-well black plate with glass bottom. (A)
4.times.10.sup.5 purified T lymphocytes (E/T=10) were added into
each well with (right) or without (left) 10 nM of CD123xCD3 BIf and
incubate at 37.degree. C., 5% CO.sub.2 overnight. Fluorescent
images were taken directly without wash. In the absence of BIf,
majority of CHO-CD123 cells were remaining attached. Conversely, in
the presence of BIf, most treated CHO-CD123 cells were detached
from the plate. (B) Same as in (A) but each well was washed twice
with PBS. The non-targeted CHO-K1 cells were not affected but most
CHO-CD123 cells were washed off.
[0042] FIG. 5A-5B. CD123xCD3 BIf-mediated T cell cellular
cytotoxicity assays. When 10 nM of CD123xCD3 BIf was used, cellular
cytotoxicities at different E/T ratio were quantitively measured by
(A) CFSE fluorescent counts of CHO-K1 (striped bars) and CHO-CD123
(open bars) cells; or (B) LDH released from CHO-K1 (striped bars)
and CHO-CD123 (open bars) cells. Total fluorescent counts in
non-treated cells; and LDH activities from Triton-lysed same number
cells were defined as 100% in (A) and (B), respectively. 10*:
E/T=10, but no BIf.
[0043] FIG. 6. The optimal E/T ratio. LDH release assays were used
to define the optimal E/T ratio in the presence (+) or absence (-)
of 10 nM CD123xCD3 BIf. CHO-K1 (striped bars) and CHO-CD123 (open
bars) cells were used as non-targeted and targeted cells. Total LDH
activities from Triton-lysed same number cells were defined as
100%.
[0044] FIG. 7. CD123xCD3 BIf dose responses. When E/T was set at 5,
different amounts of CD123xCD3 BIf were titrated into CFSE labeled
CHO-CD123 cells. 0*: in the absence of both BIf and T cells and
defined as 100%.
[0045] FIG. 8. ADCC versus T-cell cytotoxicities. 2.times.10.sup.5
PBL were added into black 96-well glass bottom plate with
4.times.10.sup.4 CFSE-labeled CHO-CD123 cells (E/T=5) and different
concentrations of 12F1scFv-Fc (striped bars) or CD123xCD3 BIf (open
bars) as indicated at X-axis. The total fluorescent counts from
un-treated cells were set as 100%; and the dotted line indicated
the activities of PBL in the absence of fusion proteins.
[0046] FIG. 9. Cytotoxic effects toward AML cell lines. AML cell
lines TF1-Hras (striped bars) and U-937 (open bars) were treated
with PBL at an E/T ratio of 5 and CD123xCD3 BIf at concentrations
indicated on the X-axis. Total LDH activities from Triton-lysed
same number cells were defined as 100%.
DESCRIPTION
[0047] A bispecific scFv immunofusion or BIf as described herein is
capable of binding cell surface molecules on a target cell, such as
cancer cells, and cell surface molecules on immune effector cell,
such as cytotoxic T lymphocytes, resulting in the targeted killing
of target cells at low effector to target ratio and/or drug
doses.
[0048] In certain aspects, the BIf targets CD123+ leukemia. One
example of a leukemia targeting BIf comprises an anti-CD123
single-chain Fv (scFv) domain fused at the N-terminus of human IgG1
hinge-C.sub.H2-C.sub.H3, and an anti-CD3 scFv fused at the
C-terminus (CD123xCD3 BIf). In certain aspects, the human IgG1 Fc
sequences can provide functional advantages. First, the Fc domain
can form a dimer that provides a more natural antibody structure.
The target cell binding affinity is between 10 to 100 fold higher
than other monomeric scFv constructs. In certain aspects, the
structural design can be a single polypeptide format that
simplifies protein production. In a further aspect, the polypeptide
can have a separate light chain and heavy chain having a chimeric
antibody like structure. Second, the polypeptides can be bound by
Fc receptors on NK cells, granulocytes and macrophage and engage
ADCC functions. Unlike traditional bispecific antibodies, the human
IgG1 Fc domain does not cause side effects associated with
immunogenicity. Third, through the salvage system, Fc region plays
a role in sustaining the antibody half-life in patient serum.
Finally, the molecular weight of approximately 140 kDa can help to
prevent kidney clearance. Thus, the polypeptides described herein
have a longer serum half-life and better tumor targeting
abilities.
[0049] Expression of the CD123xCD3 BIf in a host cell (e.g., CHO-S
cells), results in a CD123xCD3 BIf homodimer. The BIf homodimer
provides a structure of N-terminal tumor-targeting domain that
closely resembles a natural antibody. The CD123xCD3
dimeric-structure also provides binding affinity to CD123.sup.+
tumor cells with a Kd of 10.sup.-10 M, which is 1 to 2 orders of
magnitude stronger than traditional bispecific antibody constructs.
The location of the anti-CD3 scFv at C-terminus of BIf reduces the
binding affinity to CD3.sup.+ T cells by 2 orders, which helps to
prevent non-specific T cell activation. CD123xCD3 BIf is able to
achieve T cell-mediated target cell killing activities at low pM
levels with E/T ratios as low as 2. Overall, the inclusion of human
IgG1 constant region in BIf construct increases target cell binding
affinity and provides activation of additional antibody-mediated
cellular cytotoxicities.
I. Leukemia Stem Cells
[0050] Certain embodiments are directed to targeting CD123+ cancer
cells. An example of a CD123+ cell is the leukemia stem cell (LSC).
LSCs are defined as a small population of leukemia blasts with
CD34.sup.+/CD38.sup.- cell surface markers that resemble
hematopoietic stem cells (HSC) (Bonnet and Dick, 1997). LSC
isolated from leukemia patients have the capacity to repopulate
hematopoietic tissues with leukemia in NOD/SCID mice (Bonnet and
Dick, 1997; Guan and Hoggs, 2000; Guzman et al, 2001; Hope et al,
2003; Hope et al, 2004; Lapidot et al, 1994; Wang and Dick, 2005),
and possess multi-drug resistance to a variety of chemotherapeutic
agents (Costello et al, 2000; Ishikawa et al, 2007). Clinically,
poor survival has been attributed to high CD34.sup.+/CD38.sup.-
frequency at time of diagnosis in acute myeloid leukemia patients
(van Rhenen et al, 2005). It is expected that therapeutic agents
targeting LSC might be able to achieve durable remissions (Abutalib
and Tallman, 2006; Aribi et al, 2006; Morgan and Reuter, 2006; Park
et al, 2009; Stone, 2007).
[0051] The CD123 cell surface marker distinguishes LSC from
hematopoietic stem cells (HSC) (Djokic et al, 2009; Florian et al,
2006; Graf et al, 2004; Hauswirth et al, 2007; Jordan et al, 2000;
Munoz et al, 2001; Riccioni et al, 2004; Sperr et al, 2004; Testa
et al, 2002; Yalcintepe et al, 2006). CD123 is the .alpha. subunit
of interleukin-3 receptors (IL-3Rs). The function of interleukin-3
(IL-3) mediated through binding of IL-3Rs is to promote cell
survival and proliferation (Bagley et al, 1997; Blalock et al,
1999; Miyajima et al, 1993; Yen and Yang-Yen, 2006), which is one
major characteristic of cancer stem cells that make them more
resistant to conventional chemotherapeutic agents. Therapeutic
agents targeting CD123 or IL-3Rs would have the potential to
eliminate the majority of LSC, and therefore, would have a better
chance to achieve a longer disease-free survival in treated
patients.
[0052] Two approaches have been used in Phase I AML clinical trials
to target IL-3Rs. The first one is using a chimeric anti-CD123
antibody (CSL360) derived from mouse monoclonal antibody 7G3 (Jin
et al, 2009; Sun et al, 1996). Animal studies with leukemia mouse
model showed that 7G3 effectively eliminates AML-LSC through
blocking the homing effect of LSC as well as the activation of
antibody-dependent cellular cytotoxicity (ADCC) (Jin et al, 2009).
While the administration of CSL360 is safe for patients, the early
Phase I data showed minimal efficacy (Roberts et al, 2010). The
other Phase I trial is an immunotoxin, DT.sub.388IL3 (Liu et al,
2010). DT.sub.388IL3 is a fusion protein consisting of the
catalytic and translocation domains of diphtheria toxin
(DT.sub.388) fused to human IL-3 (Frankel et al, 2008; Frankel et
al, 2000; Urieto et al, 2004). Binding of IL-3 to IL-3Rs leads to
cellular internalization of DT.sub.388IL3 and results in the
activation of apoptosis and cell death (Sandvig and van Deurs,
2002; Thorburn et al, 2004). DT.sub.388IL3 was found to be
specifically cytotoxic toward AML cell lines and AML-LSC, but not
HSC (Feuring-Buske et al, 2002). DT.sub.388IL3 has been tested in
patients with refractory or relapsed AML or high-risk
myelodysplastic syndrome, and at current dose levels, no
irreversible liver or kidney toxicity was observed. There were 2
cases of complete remissions and several partial remissions (Liu et
al, 2010). Although the response rate to DT.sub.388IL3 is low, the
results did provide the proof of principle that targeting IL-3Rs is
relatively safe and could have impact on AML treatment.
[0053] Donor lymphocyte infusion has been utilized for
hematological malignancies after allogeneic stem cell
transplantation (Kolb et al, 2009; Ringden et al, 2009). The
graft-versus-leukemia activity after lymphocyte infusion has been
shown to produce durable remissions. However, these clinical
responses were only observed in a limited number of patients
(Oliansky et al, 2008). In addition, serious complications from the
graft-versus-host disease have the potential to induce morbidity
and mortality during therapy (Biagi et al, 2007). Collectively,
these clinical findings have fueled translational research for
novel therapeutic approaches to enhance the clinical benefits of
lymphocyte infusion therapy while minimizing the potential for
unwanted side effects.
[0054] Certain polypeptides described herein can be used as a
therapeutic agent to target CD123+ leukemia blasts and LSC, e.g.,
CD123xCD3 polypeptide. These novel fusion proteins belong to a
family of bispecific antibodies (Kontermann 2012; Choi et al,
2011). The 12F1 anti-CD123 murine monoclonal antibody, the highest
binding affinity obtained by Kuo et al (2009) was chosen to be used
in BIf construction. Single-chain anti-CD3 antibody was derived
from UCHT1 (Woo et al, 2008; Thompson et al, 2001; Hexham et al.,
2001) with optimized codon usage and linker design for mammalian
cell expression. Different from BiTE, these two scFvs were bridged
with a hinge-C.sub.H2-C.sub.H3 domain of human IgG1 constant
region. A similar type of construct was described before (Croasdale
et al., 2012; Dong et al, 2011; Coloma and Morrison, 1997) that
carries a natural heavy/light-chain tetrameric structure, but with
an additional scFv to a secondary antigen fused to the heavy chain
C-terminus. When expressed and secreted from a single plasmid in
suspension cultured CHO-S cells, the BIf fusion protein forms a
homo-dimer. The N- and C-terminal pairs of scFv sites with Y-shaped
binding orientations share the same Fc region as the vertical stem.
Each scFv was able to bind specific cell surface antigen and
brought cytotoxic T lymphocytes to kill targeted cells at low
effector to target ratio and drug doses.
[0055] The inclusion of human IgG1 Fc sequences in a bispecific
immunoglobulin served several functional advantages. First, the Fc
domain forms dimer that provides a more natural antibody structure
to tumor cell targeting domain. The data showed the target cell
binding affinity of CD123xCD3 BIf is about 5 times lower than the
parental antibody 12F1, but this tumor cell targeting activity is
1-2 orders of magnitude higher than other monomeric scFv constructs
(Stein et al, 2010; Hammond et al, 2007; Buhler et al, 2008; Moore
et al, 2011). Due to its natural antibody-like structure, further
humanization of CD123xCD3 to reduce immunogenicity is possible.
Second, CD123xCD3 has the potential to be bound by Fc receptors on
NK cells, granulocytes and macrophage and engage ADCC functions
(Croasdale et al., 2012). But unlike catumaxomab, the human IgG1 Fc
domain will not cause the side effects associated with
immunogenicity. Third, through FcRn-mediated salvage system, human
IgG1 Fc region could play a role in sustaining the antibody
half-life in patient serum. Besides, compared to BiTE at 50 kDa,
CD123xCD3 has a molecular weight at 140 kDa that would also help to
prevent kidney clearance. Together, it is contemplated that
CD123xCD3 will have a longer serum half life and better tumor
targeting abilities.
[0056] There is a CD123xCD16 bispecific antibody reported that
recruits NK cells for tumor cell killing (Kugler et al, 2010; Stein
et al, 2010). Compared to CSL360, CD123xCD16 brought additional
CD123.sup.+ cell killing activities. However, when PBMC was used in
their studies, the required E/T ratio was above 20. The inventors
choose to engage cytotoxic T cells for their high cytotoxic
potential, abundance, and ability to penetrate solid tumors.
Besides, cytotoxic T lymphocytes have a proven track record for
their potential to destroy malignant diseases in patients.
II. Polypeptide Compositions
[0057] Compositions of the invention include various BIfs as
described above. As used herein, an amino acid sequence or a
nucleotide sequence is "substantially the same as" or
"substantially similar to" a reference sequence if the amino acid
sequence or nucleotide sequence has at least 85% sequence identity
with the reference sequence over a given comparison window. Thus,
substantially similar sequences include those having, for example,
at least 85% sequence identity, at least 90% sequence identity, at
least 95% sequence identity or at least 99% sequence identity. Two
sequences that are identical to each other are also substantially
similar.
[0058] Sequence identity is calculated based on a reference
sequence. Algorithms for sequence analysis are known in the art,
such as BLAST, described in Altschul et al., J. Mol. Biol., 215,
pp. 403-10 (1990). In one aspect, comparisons of nucleic acid or
amino acid sequences are performed with Blast software provided by
the National Center for Biotechnology Information using a gapped
alignment with default parameters, may be used to determine the
level of identity and similarity between nucleic acid sequences and
amino acid sequences.
[0059] Mutants or variants may retain biological properties of the
bispecific immunoglobulin (SEQ ID NO:1, 2, 3, and/or 4). Mutations
or substitutions include single amino acid changes, deletions or
insertions of one or more amino acids, N-terminal truncations or
extensions, C-terminal truncations or extensions and the like. Each
polypeptide component of the bispecific immunoglobulin can be
varied in such a way as to produce a variant or mutant of the
protein defined in SEQ ID NO:1, 2, 3, and/or 4.
[0060] Mutants or variants can be generated using standard
techniques of molecular biology as described in detail in the
section "Nucleic Acid Molecules." Given the guidance provided in
the Examples, and using standard techniques, those skilled in the
art can readily generate a wide variety of additional mutants and
test whether a biological property has been altered. For example,
binding affinities or killing efficiency can be measured using in
vitro assays.
[0061] The proteins of the subject invention are present in a
non-naturally occurring environment, e.g., are recombinant or
engineered proteins. The proteins of the present invention may be
present in the isolated form, by which is meant that the protein is
substantially free of other proteins and other naturally-occurring
biological molecules, such as oligosaccharides, nucleic acids and
fragments thereof, and the like, where the term "substantially
free" in this instance means that less than 70%, usually less than
60% and more usually less than 50% of the composition containing
the isolated protein is some other natural occurring biological
molecule. In certain embodiments, the proteins are present in
substantially purified form, where by "substantially purified form"
means at least 95%, usually at least 97% and more usually at least
99% pure. In other aspects, the proteins described herein can be
expressed in vitro or in vivo, including expression in transgenic
cells or transgenic model animals.
[0062] The subject proteins may be synthetically produced, e.g., by
expressing a recombinant nucleic acid coding sequence encoding the
protein of interest in a suitable host, as described above. Any
convenient protein purification procedures may be employed, wherein
suitable protein purification methodologies are described in Guide
to Protein Purification, (Deuthser ed., Academic Press, 1990). For
example, a lysate may be prepared from the original source and
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, and the like.
III. Nucleic Acid Molecules
[0063] The present invention provides nucleic acid molecules
encoding a BIf as described herein. In one embodiment the BIf has
an amino acid sequence of SEQ ID NO:1 and can include various
mutants or variants thereof. Nucleic acid molecules encoding
shorter or longer variants of the BIf or its variants are also in
the scope of the invention.
[0064] A nucleic acid molecule as used herein is a DNA molecule,
such as a cDNA molecule, or an RNA molecule, such as an mRNA
molecule. The term "cDNA" as used herein is intended to include
nucleic acids that share the arrangement of sequence elements found
in mature mRNA species, where sequence elements are exons and 5'
and 3' non-coding regions.
[0065] Nucleic acid molecules encoding the BIf of the invention may
be synthesized from appropriate nucleotide triphosphates or
isolated from recombinant biological sources. Both methods utilize
protocols well known in the art. For example, the availability of
amino acid sequence information provided herein enables preparation
of isolated nucleic acid molecules of the invention by
oligonucleotide synthesis or by recombinant techniques. In the case
of amino acid sequence information, a number of nucleic acids that
differ from each other due to degenerate code may be synthesized.
The methods to select codon usage variants for desired hosts, such
as humans, are well known in the art.
[0066] Synthetic oligonucleotides may be prepared by the
phosphoramidite method, and the resultant constructs may be
purified according to methods known in the art, such as high
performance liquid chromatography (HPLC) or other methods as
described in, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., and under regulations described in, e.g.,
United States Dept. of HHS, National Institute of Health (NIH)
Guidelines for Recombinant DNA Research. Long, double-stranded DNA
molecules of the present invention may be synthesized by
synthesizing several smaller segments of appropriate
complementarity that comprise appropriate cohesive termini for
attachment of an adjacent segment. Adjacent segments may be linked
using DNA ligase or PCR-based methods.
[0067] Nucleic acid molecules encoding the domains and/or linkers
described herein or their equivalent may be also cloned from
biological sources or known recombinant nucleic acids.
[0068] In certain embodiments, a nucleic acid molecule of the
invention is a DNA (or cDNA) molecule comprising an open reading
frame that encodes the bispecific immunoglobulins described herein
and is capable, under appropriate conditions (e.g., cell
physiological conditions), of being expressed as a bispecific
immunoglobulin according to the invention. The invention also
encompasses nucleic acids that are homologous, substantially the
same as, identical to, or variants of the nucleic acids encoding
proteins described herein. The subject nucleic acids are
recombinant nucleic acids, i.e., they are engineered nucleic acids
not present in nature.
[0069] A nucleic acid encoding a BIf polypeptide which is an amino
acid sequence variant of the sequence shown in SEQ ID NO:1 is
further provided by the present invention. A nucleic acid encoding
such polypeptide may show greater than 60, 70, 80, 90, 95, or 99%
identity with a nucleic acid encoding SEQ ID NO: 1.
[0070] Variant nucleic acids can be generated on a template nucleic
acid selected from the described-above nucleic acids by modifying,
deleting, or adding one or more nucleotides in the template
sequence, or a combination thereof, to generate a variant of the
template nucleic acid. The modifications, additions or deletions
can be introduced by any method known in the art (see for example
Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37:
111-123; and Colicelli et al., Mol. Gen. Genet. (1985) 199:537-539,
Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989),
CSH Press, pp. 15.3-15.108) including error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-directed mutagenesis, random mutagenesis, gene reassembly,
gene site saturated mutagenesis (GSSM), synthetic ligation
reassembly (SLR), or a combination thereof. The modifications,
additions or deletions may be also introduced by a method
comprising recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation and a combination
thereof.
[0071] In addition, degenerate variants of the nucleic acids that
encode the proteins of the present invention are also provided.
Degenerate variants of nucleic acids comprise replacements of the
codons of the nucleic acid with other codons encoding the same
amino acids. In particular, degenerate variants of the nucleic
acids are generated to increase its expression in a host cell. In
this embodiment, codons of the nucleic acid that are non-preferred
or less preferred in genes in the host cell are replaced with the
codons over-represented in coding sequences in genes in the host
cell, wherein said replaced codons encode the same amino acid.
Humanized versions of the nucleic acids of the present invention
are of particular interest. As used herein, the term "humanized"
refers to changes made to the nucleic acid sequence to optimize the
codons for expression of the protein in mammalian (human) cells
(Yang et al., Nucleic Acids Research (1996) 24: 4592-4593). See
also U.S. Pat. No. 5,795,737, which describes humanization of
proteins, the disclosure of which is herein incorporated by
reference.
[0072] Also provided are vector and other nucleic acid constructs
comprising the subject nucleic acids. Suitable vectors include
viral and non-viral vectors, plasmids, cosmids, phages, etc.,
preferably plasmids, and used for cloning, amplifying, expressing,
transferring etc. of the nucleic acid sequence of the present
invention in the appropriate host. The choice of appropriate vector
is well within the skill of the art, and many such vectors are
available commercially. To prepare the constructs, the partial or
full-length nucleic acid is inserted into a vector typically by
means of DNA ligase attachment to a cleaved restriction enzyme site
in the vector. Alternatively, the desired nucleotide sequence can
be inserted by homologous recombination in vivo, typically by
attaching regions of homology to the vector on the flanks of the
desired nucleotide sequence. Regions of homology are added by
ligation of oligonucleotides, or by polymerase chain reaction using
primers comprising both the region of homology and a portion of the
desired nucleotide sequence, for example.
[0073] Also provided are expression cassettes or systems used inter
alia for the production of the subject bispecific immunoglobulin or
for replication of the subject nucleic acid molecules. The
expression cassette may exist as an extrachromosomal element or may
be integrated into the genome of the cell as a result of
introduction of said expression cassette into the cell. For
expression, the gene product encoded by the nucleic acid of the
invention is expressed in any convenient expression system,
including, for example, bacterial, yeast, insect, amphibian, or
mammalian systems. In the expression vector, a subject nucleic acid
is operatively linked to a regulatory sequence that can include
promoters, enhancers, terminators, operators, repressors and
inducers. Methods for preparing expression cassettes or systems
capable of expressing the desired product are known for a person
skilled in the art.
[0074] Cell lines, which stably express the proteins of present
invention, can be selected by the methods known in the art (e.g.,
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells that contain the gene integrated into a
genome).
[0075] The above-described expression systems may be used in
prokaryotic or eukaryotic hosts. Host-cells such as E. coli, B.
subtilis, S. cerevisiae, insect cells in combination with
baculovirus vectors, or cells of a higher organism such as
vertebrates, e.g., COS 7 cells, HEK 293, CHO, Xenopus oocytes,
primary cells etc., may be used for production or expression of the
protein.
[0076] When any of the above-referenced host cells, or other
appropriate host cells or organisms are used to replicate and/or
express the nucleic acids of the invention, the resulting
replicated nucleic acid, expressed protein or polypeptide is within
the scope of the invention as a product of the host cell or
organism. The product may be recovered by an appropriate means
known in the art.
IV. Pharmaceutical Compositions and Administration Thereof
[0077] One aspect of the invention is a pharmaceutical composition
comprising a BIf as described herein. Another aspect of the
invention is the use of bispecific antibody for the manufacture of
a pharmaceutical composition. A further aspect of the invention is
a method for the manufacture of a pharmaceutical composition
comprising a bispecific immunoglobulin as described herein. In
another aspect, the present invention provides a composition, e.g.
a pharmaceutical composition, containing a bispecific
immunoglobulin as described herein, formulated together with a
pharmaceutical carrier.
[0078] One embodiment of the invention is a bispecific
immunoglobulin as described herein for the treatment of cancer.
Another embodiment is directed to pretreating blood transfusion or
transplant products, tissues, or organs to reduce the number of
CD123+ cells in the transfusion or transplant candidate.
[0079] Another aspect of the invention is said pharmaceutical
composition for the treatment of cancer.
[0080] Another aspect of the invention is the use of a bispecific
immunoglobulin as described herein for the manufacture of a
medicament for the treatment of cancer.
[0081] Another aspect of the invention is method of treatment of
patient suffering from cancer by administering a bispecific
immunoglobulin as described herein to a patient in the need of such
treatment.
[0082] As used herein, "pharmaceutical carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for intravenous, intramuscular, subcutaneous,
parenteral, spinal or epidermal administration (e.g. by injection
or infusion).
[0083] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. To administer a compound
of the invention by certain routes of administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation. For example, the
compound may be administered to a subject in an appropriate
carrier, for example, liposomes, or a diluent. Pharmaceutically
acceptable diluents include saline and aqueous buffer solutions.
Pharmaceutical carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. The use of such
media and agents for pharmaceutically active substances is known in
the art.
[0084] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intra-arterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0085] The term cancer as used herein refers to proliferative
diseases, such as leukemias and any other cancer in which CD123+
cancer cells can be detected, including refractory versions of any
of the above cancers, or a combination of one or more of the above
cancers.
[0086] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents that delay absorption such
as aluminum monostearate and gelatin.
[0087] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0088] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient that is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0089] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier preferably is an isotonic buffered saline solution.
[0090] Proper fluidity can be maintained, for example, by use of
coating such as lecithin, by maintenance of required particle size
in the case of dispersion and by use of surfactants. In many cases,
it is preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition.
V. EXAMPLES
[0091] The following examples as well as the figures are included
to demonstrate preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the techniques
disclosed in the examples or figures represent techniques
discovered by the inventors to function well in the practice of the
invention, and thus can be considered to constitute preferred modes
for its practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many changes can
be made in the specific embodiments which are disclosed and still
obtain a like or similar result without departing from the spirit
and scope of the invention.
Example 1
CD123xCD3 BISPECIFIC IMMUNOGLOBULINS
A. Materials and Methods
Cell Lines and Antibodies
[0092] Jurkat, TF1/H-ras (Kiser et al, 2001) and U-937 cells were
maintained in RPMI medium containing 10% FBS. CHO-K1 cells (ATCC)
were maintained in ATCC-formulated F-12K medium containing 10% FBS.
Human CD123 stably transfected CHO-K1 (CHO-CD123) cells were
maintained in F-12K medium supplemented with 10% FBS and 0.4 mg/ml
zeocin (Invitrogen). FreeStyle CHO-S cells (Life
Technologies/Invitrogen) were maintained in FreeStyle CHO
Expressing medium supplemented with 8 mM L-glutamine. Hybridoma
cells producing murine anti-human CD123 antibody 12F1 was described
previously (Kuo et al, 2009).
Construction of 12F1scFv-Fc and CD123xCD3 BIf Expressing
Plasmids
[0093] Complementary DNA of 12F1 variable regions were amplified by
RT-PCR using total RNA extraction RNeasy kit (Qiagen), One-step
RT-PCR kit (Qiagen) and mouse Ig-primer sets (EMD Bioscience).
Amplified DNA fragments were cloned into linear pDrive TA cloning
vector (Qiagen) and the PCR inserts were sequenced. Correct light
chain variable (V.sub.L) and heavy chain variable (V.sub.H)
sequences were verified by comparing with mouse immunoglobulin
sequences database (AntibodyResource.com). The 12F1 scFv, in the
order of V.sub.L-218L-V.sub.H, was subsequently created by
overlapping PCR amplification of V.sub.L and V.sub.H using the
following primer pairs: P1 (GATATCGGACATTATGATGTCACAG)(SEQ ID NO:5)
and P2 (TTCACCGCTTCCCGGTTTTCCGGAGCCAGAGGTGCTACCTTTGATTTCCAGTTTG
GT)(SEQ ID NO:6); and P3
(TCCGGAAAACCGGGAAGCGGTGAAGGGTCCACCAAGGGTGTGCAGCTTCAGGA GTCG)(SEQ ID
NO:7) and P4 (AGATCTGGCTGAGGAGACTGTGAGAGT)(SEQ ID NO:8),
respectively. Primers P2 and P3 have a 24 base-pair complementary
overlap and each encoded an 18 residue 218L linker
(GSTSGSGKPGSGEGSTKG)(SEQ ID NO:9) (Whitlow et al, 1993; Vallera et
al, 2009). The 12F1scFv was then cloned into EcoRV and BglII sites
of pINFUSE-hIgG1-Fc2 vector (InvivoGen) to generate 12F1scFv-Fc
expression plasmid p12F1scFv-hIg. Codon optimized single-chain
anti-CD3 antibody sequence was derived from murine mAb UCHT1 with
an upstream linker sequence (AGATCTGGATCACCATGG)(SEQ ID NO:10),
followed by V.sub.L, 218L linker, V.sub.H and a downstream
termination codon & linker sequences (TAACTCGAGGCTAGC)(SEQ ID
NO:11). DNA was synthesized by GenScript (Piscataway, N.J.) and
cloned into the BglII and NheI sites of p12F1scFv-hIg to generate
p12F1scFv-scUCHT1. Human IgG1 hinge-C.sub.H2-C.sub.H3 fragment was
obtained by PCR with primers P5 (AGATCTGACAAAACTCACACATGC)(SEQ ID
NO:12) and P6 (CCATGGTTTACCCGGAGACAGGGA)(SEQ ID NO:13) and cloned
into BglII and NcoI sites of psc12F1-scUCHT1 to generate CD123xCD3
BIf expressing plasmid, pCD123xCD3BIf. From 5'- to 3'-ends, the
coding region sequence is:
V.sub.L(12F1)-218L-V.sub.H(12F1)-hinge-C.sub.H2-C.sub.H3-V.sub.L(UCHT1)-2-
18L-V.sub.H(UCHT1).
Expression and Purification of 12F1scFv-Fc and CD123 xCD3 BIf
[0094] Secreted 12F1scFv-Fc or CD123xCD3 BIf were expressed in
CHO-S cells transiently transfected with plasmids p12F1scFv-hIg or
pCD123xCD3BIf using TransIT-PRO transfection reagents (Mirus).
Culture supernatant was harvested 6 days post-transfection and
mixed with 0.5 ml of Protein G Sepharose beads (GE Healthcare) at
4.degree. C. for 16-18 hours. Proteins were eluted with 100 mM
triethylamine pH 11.0 and neutralized immediately with 1/10 volume
of 1M sodium phosphate pH 6.0. PD-10 desalting column (GE
Healthcare) was used to exchange the buffer of purified protein to
phosphate buffer saline (PBS) pH 7.4. Proteins were sterilized
through 0.2 .mu.m syringe filter (PALL). Protein concentrations
were measured by Bradford assays (Bio-Rad) with BSA (Pierce) as
standards and verified by reduced 12.5% SDS polyacrylamide gel
electrophoresis under reduced or non-reduced condition, and
coomassive blue R-250 staining. The protein yield is 2-5 mg/L.
Flow Cytometric Analysis
[0095] Log phase growing monolayer cells were harvested with
trypsin/EDTA solution (ATCC) and washed once with culture media and
once with PBS containing 1% BSA. 1.times.10.sup.6 cells were
resuspended in 200 .mu.l staining solution (PBS, 1% BSA, 3% normal
goat serum and indicated concentration of 12F1, 12F1scFv-Fc or
CD123xCD3 BIf) at room temperature for 1 hour. Human IgG1 (Sigma)
was used as a control. Cells were rinsed twice with PBS containing
1% BSA and incubated with PE-conjugated goat anti-human or
anti-mouse antibody (Jackson Lab) at room temperature for 1 hour.
Cells were thrice washed with PBS containing 1% BSA, resuspended in
500 .mu.l of PBS and analyzed on a Beckman Coulter Cytomic FC 500
flow cytometry.
Cell-Based Enzyme-Linked Immunosorbent Assay (CELISA)
[0096] One day prior to the experiment, 5.times.10.sup.4 CHO-K1 or
CHO-CD123 cells were seeded in each well of a flat-bottom 96-well
plate. The next day, cells were washed with PBS and fixed with 3%
formaldehyde in PBS at room temperature for 10 minutes. Fixed cells
were washed thrice with PBS containing 0.5% Tween-20 (PBST) and
incubated in Blocking Solution (PBS with 10% goat serum) for 30
minutes. A titration of 12F1, 12F1scFv-Fc or CD123xCD3 BIf was then
added at room temperature for 60 minutes, followed by washing with
PBST and then 45 minutes incubation with HRP-conjugated anti-mouse
or anti-human secondary antibodies (Jackson Lab) at room
temperature. The HRP activity was detected by HRP substrate
(R&D) for 10 minutes and stopped by 0.5N H.sub.2SO.sub.4. The
plates were read by a plate reader set at 450 nm.
Determination of Binding Affinities
[0097] For antigen on monolayer cultured cells (CHO-CD123),
cell-based enzyme-linked immunosorbent assay (CELISA, see above)
was used to evaluate binding affinities (Kuo et al, 2009). The
fusion protein or antibody concentration that has 50% of maximal
binding capacities was defined as Kd. For suspension cultured
cells, flow cytometry was used to measure binding affinities. The
fusion protein or antibody concentrations that achieve the X-axis
mean value at 50% of maximal mean value was defined as Kd.
CFSE Labeled Target Cells
[0098] CHO-K1 and CHO-CD123 cells were harvested and washed twice
with PBS and resuspended in 1 ml of PBS. Two .mu.l of freshly
prepared CellTrace (Invitrogen) CFSE (5 mM in DMSO) were added into
cell suspension and incubate at 37.degree. C. for 15 minutes.
Labeled cells were washed twice with PBS, resuspended in culture
medium and seeded in black 96-well glass-bottom plate (Costar) at
4.times.10.sup.4 cell/well for cellular cytotoxicity assays.
Isolation of Peripheral Blood Lymphocytes and T Lymphocytes
[0099] Human peripheral blood mononuclear cells (PBMCs) were
isolated from fresh buffy coats of consented healthy donors using
Ficoll-Paque Premium gradient centrifugation solution (GE
Healthcare) or purchased from Astarte Biologics (Redmond, Wash.).
After overnight incubation at 37.degree. C., 5% CO.sub.2 in RPMI
medium containing 10% heat-inactivated FBS and 100 units/ml of
recombinant human IL-2 (eBioscience), the nonadherent fraction of
PBMCs was collected and used as peripheral blood lymphocytes
(PBL).
[0100] Human T lymphocytes were enriched from fresh blood of
consented donors using RosetteSep HLA enrichment cocktail (StemCell
Technologies) and cultured at 37.degree. C., 5% CO.sub.2 in RPMI
medium containing 10% heat-inactivated FBS and 100 units/ml of
recombinant human IL-2.
Cellular Cytotoxicity Assays
[0101] Cytotoxicity was measured in a standard colorimetric
quantitative lactate dehydrogenase (LDH) release measurement assay
(Promega) following manufactural instruction. Briefly,
4.times.10.sup.4 target cells were mixed with different amounts of
12F1scFv-Fc or CD123xCD3 BIf and T lympnocyte fraction at indicated
effector to target (E/T) ratio. Cells were incubated at 37.degree.
C. and 5% CO.sub.2 for 18-24 hours. After centrifugation, 50 .mu.l
of clear supernatant from each well was withdrawn, transferred to
another 96-well plate and mixed with 50 .mu.l of LDH assay
substrate at room temperature for 30 minutes. LDH activities
released from the same number of target cells treated with Triton
Lysis Solution, after subtracting the targeted cell spontaneous LDH
release, was defined as 100% lysis activity.
[0102] For CHO-K1 and CHO-CD123 cells, toxicity assays were also
tested in CFSE-labeled target cells. Labeled cells were seeded in
black 96-well plates with glass bottoms for 4-6 hours to ensure
proper attachment. Fusion proteins and effector cells were added at
indicated amounts and incubated at 37.degree. C. and 5% CO.sub.2
for 18-24 hours. Cells were washed twice with PBS. CFSE fluorescent
counts in remaining cells were measured with a GloMax fluorescent
plate reader (Promega). The fluorescence reading of mock-treated
target cells were defined as 100%. Images were also taken by
fluorescent microscope and labeled by Adobe Photoshop.
B. Results
Construction of 12F1scFv-Fc and CD123xCD3 BIf
[0103] Predicted protein structures of 12F1scFv-Fc and CD123xCD3
BIf were shown in FIGS. 1A and 1B. The only difference between
these two fusion proteins is that BIf carries the additional
C-terminal single-chain anti-CD3 antibody sequences. When
transiently expressed from CHO-S cells, 12F1scFv-Fc had a higher
yield at 5 mg/L versus CD123xCD3 BIf at 2 mg/L. Single step Protein
G Sepharose purification yields high purities of both fusion
proteins (FIG. 1C).
Cell Surface CD123 Binding Activities
[0104] The abilities of 12F1scFv-Fc and CD123xCD3 BIf to bind cell
surface CD123 were examined by flow cytometry using CHO-K1 and
CHO-CD123. 12F1 murine monoclonal antibody was used as a positive
control. At 1 .mu.g/ml, all three proteins showed strong binding
activities to CHO-CD123 cells (FIGS. 2A to 2C), but not CHO-K1
cells (data not shown). The binding affinities were tested in
cell-based ELISA with CHO-CD123 cells. The 12F1scFv-Fc and
CD123xCD3 BIf showed lower binding affinities than 12F1 (FIG. 2D),
but the Kd values are still at between 10.sup.-11 and 10.sup.-1M, a
target-cell affinity that is 1-2 orders higher than any currently
developed bispecific antibodies with monomeric scFv (Stein et al,
2010; Hammond et al, 2007; Buhler et al, 2008; Moore et al,
2011).
T Cell Binding Activities
[0105] Jurkat T cells were used to test CD123xCD3 BIf's abilities
to recruit T cells. At 1 .mu.g/ml, CD123xCD3 BIf already showed
lower binding activities than the original UCHT1 antibody to Jurkat
cells in flow cytometric analysis (FIGS. 3A and 3B). When serial
titration of antibodies and the mean X-axis values were used to
measure binding affinities, CD123xCD3 BIf showed a Kd at
10.sup.-8M, 2 orders higher than that of UCHT1 (FIG. 3C). This
reduced T cell binding affinity would help to prevent non-specific
T-cell activation.
BIf-Mediated T Cell Cytotoxicities
[0106] Since our BIf construct contains the Fc region of human IgG1
sequences, it is possible to be bound by Fc receptors on effector
cells and activate ADCC. To focus only on T cell cytotoxicity,
total T lymphocytes purified from healthy donors were used to test
BIf activities. Two assays were conducted to evaluate BIf and
effector cells efficacies. Target cell released lactose
dehydrogenase (LDH) during cell lysis has been reported to test
effector functions. The higher LDH activities represent the higher
cytotoxicities. The inventors also used CFSE pre-labeled adherent
cells as targets and compared the remaining fluorescent counts
after treatments. The lower remaining fluorescent counts indicate
stronger cellular cytotoxicities. These two methods showed
comparable results in all tests.
[0107] To begin, 10 nM of BIf was used to test the optimal
effector/target ratio (E/T). Initial tests showed that the efficacy
reach plateau at an E/T ratio less than 10 (FIG. 4 and data not
shown), and further titrations were done to refine the optimal E/T
ratio. As shown in FIG. 5, the optimal E/T ratio is at between 3
and 10. The inventors found that, in the absence of BIf (FIGS. 5A
and 5B, 10*), the T-cell alone had cell lysis activities (compare 0
and 10*). To focus on the BIf effect, reactions at even more
narrowed E/T ratio ranges with or without BIf were compared.
Indeed, the non-specific T cell killing effect is dose-dependent,
and the additional 10 nM BIf dramatically enhanced the cytotoxic
effects in CHO-CD123, but not in non-targeted CHO-K1 cells (FIG.
6). The target-specific cellular cytotoxicity can be seen at an E/T
ratio as low as 2.
[0108] The E/T ratio was fixed at 5 to test BIf dose responses. As
shown in FIG. 7, the effective dose to achieve 50% maximal cellular
cytotoxicity is at .about.10.sup.-11M, and the maximal activity can
be reached at the concentration of 10.sup.-10M. It is interesting
to note that in repeated tests, BIf at higher than optimal
concentrations produced less cytotoxic effects.
T Cell Cytotoxicity Versus ADCC
[0109] As mentioned earlier, the included human IgG1 Fc region in
our BIf construct could also interact with other effector cells
carrying Fc receptors and activate ADCC responses. To distinguish
ADCC and T cell cytotoxicity, the non-adherent peripheral blood
lymphocytes (PBL) were used as effector cells, and 12F1scFv-Fc was
used as ADCC control. CFSE-labeled CHO-CD123 cells were treated
with a serial titration of 12F1scFv-Fc or CD123xCD3 BIf and PBL at
an E/T ratio of 5 for overnight. The remaining fluorescent counts
after washes were measured by a plate reader. While 12F1scFv-Fc
started to induce ADCC at .about.200 pM and showed low cytotoxic
effects at 5 nM, CD123xCD3 BIf showed stronger cytotoxic effects at
low pM levels and reached a maximum at 200 pM (FIG. 8). The
inventors concluded that ADCC might contribute to BIf's target cell
killing activities, but in a very minor role.
Cytotoxic Effects Toward AML Cell Lines
[0110] Established AML cell lines were used as target cells to test
BIf efficacies. TF1 cells transfected with H-ras were used
extensively in our previous studies targeting IL-3Rs and the
estimated average CD123 copy number on these cells is less than
1,000. Compared to CHO-CD123 cells that have average 30,000 to
60,000 copies per cell, TF1/Hras is considered to be low on CD123
copy number. U-937 was also reported as a CD123+ AML cell line with
a lower number of cell surface CD123. These suspension cultured
cells were tested in BIf-mediated T cell cytotoxicity assays using
released LDH as an indicator. As shown in FIG. 9, TF1/Hras is
sensitive to BIf at around the same concentration as CHO-CD123 and
U-937 required slightly higher concentrations to reach the same
efficacies. The inventors concluded that CD123xCD3 BIf carries the
activities to bring CD123+ AML cells and CD3+ cytotoxic T
lymphocyte together to achieve target cell killing activities.
Sequence CWU 1
1
191726PRTArtificial SequenceSynthetic peptide 1Asp Ile Met Met Ser
Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Phe
Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Phe Phe Gly 20 25 30 Ser
Thr Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Ala 65 70 75 80 Ile Ser Ser Val Met Pro Glu Asp Leu Ala Val Tyr
Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly
Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 130 135 140 Ser Gln Ser Leu
Ser Leu Thr Cys Ser Val Thr Asp Tyr Ser Ile Thr 145 150 155 160 Ser
Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 165 170
175 Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro
180 185 190 Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
Asn Gln 195 200 205 Phe Phe Leu Lys Leu Ser Ser Val Thr Thr Glu Asp
Thr Ala Thr Tyr 210 215 220 Tyr Cys Ser Arg Gly Glu Gly Phe Tyr Phe
Asp Ser Trp Gly Gln Gly 225 230 235 240 Thr Thr Leu Thr Val Ser Ser
Ala Arg Ser Asp Lys Thr His Thr Cys 245 250 255 Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 260 265 270 Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 275 280 285 Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 290 295
300 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
305 310 315 320 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu 325 330 335 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 340 345 350 Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys 355 360 365 Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 370 375 380 Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 385 390 395 400 Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 405 410 415
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 420
425 430 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln 435 440 445 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn 450 455 460 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys Pro Trp Asp 465 470 475 480 Ile Gln Met Thr Gln Thr Thr Ser
Ser Leu Ser Ala Ser Leu Gly Asp 485 490 495 Arg Val Thr Ile Ser Cys
Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu 500 505 510 Asn Trp Tyr Gln
Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr 515 520 525 Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser 530 535 540
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 545
550 555 560 Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
Trp Thr 565 570 575 Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser
Thr Ser Gly Ser 580 585 590 Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
Lys Gly Glu Val Gln Leu 595 600 605 Gln Gln Ser Gly Pro Glu Leu Val
Lys Pro Gly Ala Ser Met Lys Ile 610 615 620 Ser Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp 625 630 635 640 Val Lys Gln
Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn 645 650 655 Pro
Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala 660 665
670 Thr Phe Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu
675 680 685 Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
Ser Gly 690 695 700 Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp
Gly Ala Gly Thr 705 710 715 720 Thr Val Thr Val Ser Ser 725
2250PRTArtificial SequenceSynthetic peptide 2Asp Ile Met Met Ser
Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Phe
Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Phe Phe Gly 20 25 30 Ser
Thr Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Ala 65 70 75 80 Ile Ser Ser Val Met Pro Glu Asp Leu Ala Val Tyr
Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gly Ser Thr Ser Gly Ser Gly
Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 130 135 140 Ser Gln Ser Leu
Ser Leu Thr Cys Ser Val Thr Asp Tyr Ser Ile Thr 145 150 155 160 Ser
Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 165 170
175 Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro
180 185 190 Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
Asn Gln 195 200 205 Phe Phe Leu Lys Leu Ser Ser Val Thr Thr Glu Asp
Thr Ala Thr Tyr 210 215 220 Tyr Cys Ser Arg Gly Glu Gly Phe Tyr Phe
Asp Ser Trp Gly Gln Gly 225 230 235 240 Thr Thr Leu Thr Val Ser Ser
Ala Arg Ser 245 250 3247PRTArtificial SequenceSynthetic peptide
3Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1
5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe
Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Ala Gly Gly
Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys
Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln 115 120 125 Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys 130 135
140 Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn
145 150 155 160 Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met
Gly Leu Ile 165 170 175 Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln
Lys Phe Lys Asp Lys 180 185 190 Ala Thr Phe Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr Met Glu Leu 195 200 205 Leu Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys Ala Arg Ser 210 215 220 Gly Tyr Tyr Gly Asp
Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly 225 230 235 240 Thr Thr
Val Thr Val Ser Ser 245 4229PRTArtificial Sequencesynthetic peptide
4Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1
5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135
140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Pro Trp
225 525DNAArtificial SequenceSynthetic oligonucleotide 5gatatcggac
attatgatgt cacag 25657DNAArtificial SequenceSynthetic
oligonucleotide 6ttcaccgctt cccggttttc cggagccaga ggtgctacct
ttgatttcca gtttggt 57757DNAArtificial SequenceSynthetic
oligonucleotide 7tccggaaaac cgggaagcgg tgaagggtcc accaagggtg
tgcagcttca ggagtcg 57827DNAArtificial SequenceSynthetic
oligonucleotide 8agatctggct gaggagactg tgagagt 27918PRTArtificial
SequenceSynthetic peptide 9Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly
Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly 1018DNAArtificial
SequenceSynthetic oligonucleotide 10agatctggat caccatgg
181115DNAArtificial SequenceSynthetic oligonucleotide 11taactcgagg
ctagc 151224DNAArtificial Sequencesynthetic oligonucleotide
12agatctgaca aaactcacac atgc 241324DNAArtificial SequenceSynthetic
oligonucleotide 13ccatggttta cccggagaca ggga 2414114PRTArtificial
SequenceSynthetic polypeptide 14Asp Ile Val Met Thr Gln Thr Pro Pro
Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys
Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr
Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu
Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Arg Ala 15116PRTArtificial
SequenceSynthetic polypeptide 15Gln Val Lys Leu Glu Glu Ser Gly Ala
Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val
Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile
Ser Thr Tyr Phe Gly Asp Ala Thr Tyr Asn Gln Lys Phe 50 55 60 Lys
Gly Lys Ala Thr Met Thr Val Asp Ser Ser Ser Thr Ala Tyr Met 65 70
75 80 Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
Ala 85 90 95 Arg Glu Asp Tyr Val Pro Phe Ala Tyr Trp Gly Gln Gly
Thr Leu Val 100 105 110 Thr Val Ser Ala 115 16108PRTArtificial
SequenceSynthetic polypeptide 16Trp Asp Ile Val Met Thr Gln Ser His
Lys Phe Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Ile Ile
Cys Lys Ala Ser Gln Asp Val Gly Thr 20 25 30 Ala Val Asp Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr 50 55 60 Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln 65 70
75 80 Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr
Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys 100
105 17115PRTArtificial SequenceSynthetic polypeptide 17Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Thr 1 5 10 15 Ser
Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25
30 Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45 Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln
Lys Phe 50 55 60 Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Asn Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115
18107PRTArtificial SequenceSynthetic polypeptide 18Asp Ile Gln Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Val 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Asn Ser Ser Ile Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg 100 105 19117PRTArtificial SequenceSynthetic polypeptide
19Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp
Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Val Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser
Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Trp Ala Asp Ala
Phe Asp Ile Trp Gly Glu Gly Thr Met 100 105 110 Val Thr Val Ser Ser
115
* * * * *