U.S. patent application number 17/248174 was filed with the patent office on 2021-05-06 for trifunctional t cell-antigen coupler and methods and uses thereof.
The applicant listed for this patent is McMaster University. Invention is credited to Jonathan BRAMSON, Galina DENISOVA, Rajanish GIRI, Christopher W. HELSEN, Kenneth MWAWASI.
Application Number | 20210130435 17/248174 |
Document ID | / |
Family ID | 1000005341435 |
Filed Date | 2021-05-06 |
![](/patent/app/20210130435/US20210130435A1-20210506\US20210130435A1-2021050)
United States Patent
Application |
20210130435 |
Kind Code |
A1 |
BRAMSON; Jonathan ; et
al. |
May 6, 2021 |
TRIFUNCTIONAL T CELL-ANTIGEN COUPLER AND METHODS AND USES
THEREOF
Abstract
A trifunctional molecule comprising a target-specific ligand, a
ligand that binds a protein associated with the TCR complex and a T
cell receptor signaling domain polypeptide is provided Engineering
T cells with this novel receptor engenders antigen specific
activation of numerous T cell functions, including cytokine
production, degranulation and cytolysis.
Inventors: |
BRAMSON; Jonathan;
(Oakville, CA) ; HELSEN; Christopher W.;
(Oakville, CA) ; DENISOVA; Galina; (Hamilton,
CA) ; GIRI; Rajanish; (Telliarganj Allahabad, IN)
; MWAWASI; Kenneth; (Hamilton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
McMaster University |
Hamilton |
|
CA |
|
|
Family ID: |
1000005341435 |
Appl. No.: |
17/248174 |
Filed: |
January 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15929510 |
May 6, 2020 |
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17248174 |
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16547421 |
Aug 21, 2019 |
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15929510 |
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15117173 |
Aug 5, 2016 |
10435453 |
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PCT/CA2015/000068 |
Feb 6, 2015 |
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16547421 |
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61936906 |
Feb 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/17 20130101;
C07K 16/32 20130101; C07K 2317/622 20130101; C07K 2319/03 20130101;
C07K 2317/31 20130101; C07K 2319/00 20130101; C07K 16/2809
20130101; C07K 14/70514 20130101; C07K 2317/73 20130101; C07K
2318/20 20130101; C07K 14/7051 20130101 |
International
Class: |
C07K 14/73 20060101
C07K014/73; A61K 35/17 20060101 A61K035/17; C07K 16/28 20060101
C07K016/28; C07K 14/725 20060101 C07K014/725; C07K 16/32 20060101
C07K016/32 |
Claims
1. A T cell comprising a nucleic acid encoding a T cell Antigen
Coupler (TAC), the nucleic acid comprising: a) a first
polynucleotide encoding an antigen binding domain which binds a
tumor antigen; b) a second polynucleotide encoding a ligand that
binds a CD3 protein associated with a TCR complex on the T cell,
wherein the second polynucleotide comprises a sequence as set forth
in SEQ ID NO: 13 or SEQ ID NO: 24; and c) a third polynucleotide
encoding a T cell co-receptor domain polypeptide wherein the T cell
co-receptor domain polypeptide comprises a CD4 cytosolic domain and
a CD4 transmembrane domain; wherein the components (a), (b) and (c)
are fused directly to each other or joined by at least one linker,
and provided that the nucleic acid does not encode a co-stimulatory
domain.
2. The T cell of claim 1, wherein the first polynucleotide
comprises a sequences as set forth in SEQ ID NO: 7 or SEQ ID NO:
22.
3. The T cell of claim 1, wherein the antigen binding domain binds
to human epidermal growth factor receptor 2 (HER2).
4. The T cell of claim 1, wherein the antigen binding domain binds
to B-cell maturation antigen (BCMA).
5. A pharmaceutical composition comprising the T cell of claim 1,
and a carrier.
6. A method of treating a cancer that expresses a tumor antigen in
an individual in need thereof, the method comprising administering
the T cell of claim 1 to the individual, wherein the antigen
binding domain binds to the tumor antigen expressed by the cancer,
thereby treating the cancer.
7. A T cell comprising a nucleic acid encoding a T cell Antigen
Coupler (TAC), the nucleic acid comprising: a) a first
polynucleotide encoding an antigen binding domain which binds a
tumor antigen; b) a second polynucleotide encoding a ligand that
binds a CD3 protein associated with a TCR complex on the T cell;
and c) a third polynucleotide encoding a T cell co-receptor domain
polypeptide wherein the T cell co-receptor domain polypeptide
comprises a CD4 cytosolic domain and a CD4 transmembrane domain,
wherein the third polynucleotide comprises a sequence as set forth
in SEQ ID NO: 17; wherein the components (a), (b) and (c) are fused
directly to each other or joined by at least one linker, and
provided that the nucleic acid does not encode a co-stimulatory
domain.
8. The T cell of claim 7, wherein the ligand that binds the CD3
protein associated with the TCR complex on the T cell is UCHT1.
9. The T cell of claim 7, wherein the first polynucleotide
comprises a sequence as set forth in SEQ ID NO: 7 or SEQ ID NO:
22.
10. The T cell of claim 7, wherein the antigen binding domain binds
to human epidermal growth factor receptor 2 (HER2).
11. The T cell of claim 1, wherein the antigen binding domain binds
to B-cell maturation antigen (BCMA).
12. A pharmaceutical composition, comprising the T cell of claim 7,
and a carrier.
13. A method of treating a cancer that expresses a tumor antigen in
an individual in need thereof, the method comprising administering
the T cell of claim 7 to the individual, wherein the antigen
binding domain binds to the tumor antigen expressed by the cancer,
thereby treating the cancer.
14. A T cell comprising a nucleic acid encoding a T cell Antigen
Coupler (TAC), the nucleic acid comprising: a) a first
polynucleotide encoding an antigen binding domain which binds a
tumor antigen; b) a second polynucleotide encoding a ligand that
binds a CD3 protein associated with a TCR complex on the T cell,
wherein the second polynucleotide comprises a sequence as set forth
in SEQ ID NO: 13 or SEQ ID NO: 24; and c) a third polynucleotide
encoding a T cell co-receptor domain polypeptide wherein the T cell
co-receptor domain polypeptide comprises a CD4 cytosolic domain and
a CD4 transmembrane domain, wherein the third polynucleotide
comprises a sequence as set forth in SEQ ID NO: 17; wherein the
components (a), (b) and (c) are fused directly to each other or
joined by at least one linker, and provided that the nucleic acid
does not encode a co-stimulatory domain.
15. The T cell of claim 14, wherein the first polynucleotide
comprises a sequence as set forth in SEQ ID NO: 7 or SEQ ID NO:
22.
16. The T cell of claim 14, wherein the antigen binding domain
binds to human epidermal growth factor receptor 2 (HER2).
17. The T cell of claim 14, wherein the antigen binding domain
binds to B-cell maturation antigen (BCMA).
18. A pharmaceutical composition, comprising the T cell of claim
14, and a carrier.
19. A method of treating a cancer that expresses a tumor antigen in
an individual in need thereof, the method comprising administering
the T cell of claim 14 to the individual, wherein the antigen
binding domain binds to the tumor antigen expressed by the cancer,
thereby treating the cancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/929,510, filed on May 6, 2020, which is a continuation of
application Ser. No. 16/547,421, filed on Aug. 21, 2019, which is a
continuation of application Ser. No. 15/117,173, filed on Aug. 5,
2016, now issued as U.S. Pat. No. 10,435,453, which is a national
stage entry of Internal Application No. PCT/CA2015/000068 filed on
Feb. 6, 2015, which claims priority to U.S. Provisional Patent
Application No. 61/936,906 filed on Feb. 7, 2014, the contents of
which are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 12, 2021, is named TMV_001C3_SL.txt and is 31,075 bytes in
size.
FIELD
[0003] The present disclosure relates to a method of treating
cancer by engineering T-cells with high cytotoxicity against
specific target cells and reduced off-target toxicity. In
particular, the disclosure relates to engineering T-cells to
express novel biological agents which mimic the natural T-cell
activation process.
BACKGROUND
[0004] Cancer is a major health challenge, with over 150,000 cases
of cancer expected to be diagnosed in Canada in 2013 alone. While
patients with early stage disease can be treated effectively by
conventional therapies (surgery, radiation, chemotherapy), few
options are available to patients with advanced disease and those
options are typically palliative in nature. Active immunotherapy
seeks to employ the patient's immune system to clear tumor deposits
and offers an exciting option to patients who have failed
conventional therapies (Humphries, 2013). Indeed, several clinical
studies have demonstrated that immunotherapy with T cells can be
curative in patients with advanced melanoma, confirming the utility
of this approach (Humphries, 2013). Additionally, patients
suffering from chronic lymphocytic leukemia (CLL) and acute
lymphoblastic leukemia (ALL) have also been effectively treated and
cured with T cell immunotherapy (Fry and Mackall,
2013)(Kochenderfer and Rosenberg, 2013). While there are several
immunotherapy approaches, the engineering of T cells with chimeric
receptors allows any patient's immune cells to be targeted against
any desirable target in a major histocompatibility complex (MHC)
independent manner. To date, the chimeric receptors used for
engineering T cells consist of a targeting domain, usually a
single-chain fragment variable (scFv); a transmembrane domain; and
a cytosolic domain that contains signaling elements from the T cell
receptor and associated proteins (Dotti et al., 2009). Such
chimeric receptors have been referred to as "T-body", "Chimeric
Antigen Receptor" (CAR) or "Chimeric Immune Receptor"
(CIR)--currently, most researchers use the term "CAR" (Dotti et
al., 2009). These CARs are considered in modular terms and
scientists have spent considerable time investigating the influence
of different cytoplasmic signaling domains on CAR function. The
first-generation CARs employed a single signaling domain from
either CD3.zeta. or FccRl.gamma.. Second-generations CARs combined
the signaling domain of CD3.zeta. with the cytoplasmic domain of
costimulatory receptors from either the CD28 or TNFR family of
receptors (Dotti et al., 2009). Third-generation CARs combined
multiple costimulatory domains, but there is concern that
third-generation CARs may lose antigen-specificity (Han et al.,
2013). Most CAR-engineered T cells that are being tested in the
clinic employ second-generation CARs where CD3.zeta. is coupled to
the cytoplasmic domain of either CD28 or CD137 (Han et al.,
2013)(Finney et al., 2004)(Milone et al., 2009).
[0005] While CAR-engineered T cells have shown considerable promise
in clinical application, they rely on a synthetic method for
replacing the activation signal that is provided by the T cell
receptor (TCR). Since this synthetic receptor does not deliver all
of the signaling components associated with the TCR (ex.
CD3epsilon, Lck), it remains unclear whether the T cells are
optimally activated by the CAR or how the CAR activation affects T
cell differentiation (ex. progression to memory). Furthermore,
since the CAR signaling domains are disconnected from their natural
regulatory partners by the very nature of the CAR structure, there
is also an inherent risk that CARs may lead to a low-level of
constitutive activation which could result in off-target
toxicities.
[0006] Given these limitations, it is preferable to re-direct T
cells to attack tumors via their natural TCR. To this end, a class
of recombinant proteins termed "Bispecific T-cell Engagers" (BiTEs)
has been created (Chames and Baty, 2009)(Portell et al., 2013).
These proteins employ bispecific antibody fragments to crosslink
T-cell TCR receptors with target antigens. This leads to efficient
T-cell activation, triggering cytotoxicity. Similarly, bi-specific
antibodies have been generated that accomplish this goal and some
scientists have simply linked anti-CD3 antibodies to tumor-specific
antibodies employing chemical linkage (Chames and Baty, 2009).
While these bi-specific proteins have demonstrated some activity in
vitro, GMP production, short biological half-lives and
bioavailability represent significant challenges to the successful
use of these molecules in cancer treatment. Additionally, these
molecules also fail to properly recapitulate natural TCR signaling
because they do not engage the TCR co-receptors (CD8 and CD4).
[0007] Accordingly, a need remains for T cell-antigen couplers with
enhanced activity and safety compared to traditional CARs.
SUMMARY
[0008] The present inventors have demonstrated that a trifunctional
T cell-antigen coupler that better mimics the natural signaling
through the T cell receptor (TCR), while retaining major
histocompatibility complex unrestricted targeting, has enhanced
activity and safety compared to traditional chimeric antigen
receptors.
[0009] Accordingly, one aspect of the disclosure provides a nucleic
acid comprising: [0010] a. a first polynucleotide encoding a
target-specific ligand; [0011] b. a second polynucleotide encoding
a ligand that binds a protein associated with the TCR complex; and
[0012] c. a third polynucleotide encoding a T cell receptor
signaling domain polypeptide.
[0013] Another aspect of the disclosure provides a polypeptide
encoded by the nucleic acid described above.
[0014] Another aspect of the disclosure provides an expression
vector comprising the nucleic acid described above.
[0015] Yet another aspect of the disclosure provides a T-cell
expressing the nucleic acid described above. Another aspect of the
disclosure provides a pharmaceutical composition comprising the T
cell and a carrier.
[0016] The disclosure also provides a use of a T cell for treating
cancer in a subject in need thereof, wherein the T cell expresses a
nucleic acid comprising: [0017] a. a first polynucleotide encoding
a target-specific ligand; [0018] b. a second polynucleotide
encoding a ligand that binds a protein associated with the TCR
complex; and [0019] c. a third polynucleotide encoding a T cell
receptor signaling domain polypeptide.
[0020] In one embodiment, the target-specific ligand binds an
antigen on a cancerous cell.
[0021] In another embodiment, the target-specific ligand is a
designed ankyrin repeat (DARPin) polypeptide or scFv.
[0022] In another embodiment, the protein associated with the TCR
complex is CD3.
[0023] In another embodiment, the ligand that binds a protein
associated with the TCR complex is a single chain antibody.
[0024] In another embodiment, the ligand that binds a protein
associated with the TCR complex is UCHT1, or a variant thereof.
[0025] In another embodiment, the T cell receptor signaling domain
polypeptide comprises a cytosolic domain and a transmembrane
domain.
[0026] In another embodiment, the cytosolic domain is a CD4
cytosolic domain and the transmembrane domain is a CD4
transmembrane domain.
[0027] In another embodiment the first polynucleotide and third
polynucleotide are fused to the second polynucleotide.
[0028] In another embodiment, the second polynucleotide and third
polynucleotide are fused to the first polynucleotide.
[0029] The disclosure also provides a vector construct comprising:
[0030] a. a first polynucleotide encoding a target-specific ligand;
[0031] b. a second polynucleotide encoding a ligand that binds a
protein associated with the TCR complex; and [0032] c. a third
polynucleotide encoding a T cell receptor signaling domain
polypeptide, and [0033] d. a promoter functional in a mammalian
cell.
[0034] In one embodiment, the first polynucleotide and third
polynucleotide are fused to the second polynucleotide to provide a
T cell antigen coupler fusion and the coding sequence of the T cell
antigen coupler fusion is operably connected to the promoter.
[0035] In another embodiment, the second polynucleotide and third
polynucleotide are fused to the first polynucleotide to provide a T
cell antigen coupler fusion and the coding sequence of the T cell
antigen coupler fusion is operably connected to the promoter.
[0036] The disclosure also provides an isolated T cell transfected
with the vector construct.
[0037] Other features and advantages of the present disclosure will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples while indicating preferred embodiments of the
disclosure are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
disclosure will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A-D is a graphic summary of the trifunctional T
cell-antigen coupler (Tri-TAC) in comparison with a conventional
second-generation CAR. A schema of the constructs used in this work
is included.
[0039] FIG. 2 shows a surface expression analysis of the Tri-TAC
variants and the classical CAR.
[0040] FIGS. 3A-B show an analysis of cell activation looking at
the different markers IFN-.gamma., TNF-.alpha. and CD107a.
[0041] FIG. 4 analyzes the killing of two different cell lines that
either express (D2F2E2) or do not express (D2F2) the molecular
target of the classical CAR and the Tri-TAC.
[0042] FIGS. 5A-D depict natural T-cell initiation (5A), two
currently used artificial methods for T-cell activation (5B and
5C), and the TAC activation technology (5D).
[0043] FIGS. 6A-B depicts (6A) configuration 1 of the TAC molecule
and (6B) configuration 2 of the TAC molecule.
[0044] FIGS. 7A-C show the functionality of scFv CD4 TAC. (7A) is a
histogram showing surface expression of the scFv CD4 TAC receptor
relative to empty vector, (7B) shows antigen specific activation of
T-cells expressing either the scFv CD4 TAC (top) or scFV CAR
(bottom) and (7C) shows comparable killing of MCF-7 human tumour
cell line (Her2 positive) by both scFv CD4 TAC and scFv CAR.
[0045] FIGS. 8A-C characterize the CD4-TAC configuration 2. (8A) is
a histogram showing surface expression of the DARPin CD4 TAC
receptor relative to empty vector, (8B) shows cytokine production
and degranulation of T cell engineered with DARPin TAC
configuration 2 exposed to Her2 antigen and (8C) shows growth of
CD4 TAC configuration 2 relative to empty vector control.
[0046] FIGS. 9A-D shows the functionality of DARPin CD4 TAC
configuration 1. (9A) shows the surface expression of DARPin CD4
YAC compared to DARPin CAR and the NGFR only control, (9B) shows
growth of CD4 TAC configuration 1 and (9C) and (9D) show the
percentage of cells positive for various activation and degradation
markers.
[0047] FIG. 10 shows the cytotoxicity and overall activity of TAC
and CAR. Cells engineered with TAC, CAR or empty vector control
were incubated in various human tumor cell lines.
[0048] FIGS. 11A-B show receptor surface expression and activation
of various TAC controls. (11A) shows cell surface expression
(left), degranulation (middle) and cytokine production (right) and
(11B) shows that only full length CD4-TAC is able to elicit a
cytotoxic response.
[0049] FIGS. 12A-C show properties of various transmembrane TAC
variants. (12A) is an overview of various transmembrane domain
constructs, (12B) shows the surface of expression of various
constructs engineered in CD8 purified T cells and (12C) shows
testing of the various variants for degranulation and cytokine
production.
[0050] FIG. 13 shows Lck interaction with TAC variants. The top
panel shows the ability of full length TAC and the cytosolic
deletion to pull down Lck and the bottom panel is a densitometry
analysis of Lck detected in the pellets of.
[0051] FIGS. 14A-B show CD4 TAC surface expression and activity
compared to a BiTE like variant. (14A) depicts surface expression
of an NGFR only control, CD4 TAC and BiTE like variant and (14B)
compares cytotoxicity in various cell lines.
[0052] FIGS. 15A-C shows wild type CD4 TAC compared to a random
mutagen library of UCHT1. (15A) shows the schematic representation
of the mutant, (15B) is a histogram showing surface expression of
the library and (15C) shows the ability of the library to activate
T cells and produce cytokines.
[0053] FIGS. 16A-C shows enhanced surface expression of the A85V,
T161P mutant. (16A) compares final CD/CD8 populations between CD4
TAC and A85V, T161P mutant, (16B) shows enhanced surface expression
of the A85V, T161P mutant and (16C) shows that cytokine production
is diminished in the A85V, T161 mutant.
[0054] FIGS. 17A-B shows A85V, T161P mutant cytotoxicity and
growth. (17A) shows cytotoxicity of the A85V, T161P mutant in
various cell lines and (17B) shows cell growth in culture over 2
weeks.
DETAILED DESCRIPTION
(i) Definitions
[0055] The term "a cell" as used herein includes a single cell as
well as a plurality of cells.
[0056] The term "T cell" as used herein refers to a type of
lymphocyte that plays a central role in cell-mediated immunity. T
cells, also referred to as T lymphocytes, can be distinguished from
other lymphocytes, such as B cells and natural killer cells, by the
presence of a T-cell receptor (TCR) on the cell surface. There are
several subsets of T cells with distinct functions, including but
not limited to, T helper cells, cytotoxic T cells, memory T cells,
regulatory T cells and natural killer T cells.
[0057] The term "T cell antigen coupler" as used herein refers to
an engineered nucleic acid construct or polypeptide, that when
expressed on a T cell, targets the T cell to a particular
antigen.
[0058] The term "polynucleotide" and/or "nucleic acid sequence"
and/or "nucleic acid" as used herein refers to a sequence of
nucleoside or nucleotide monomers consisting of bases, sugars and
intersugar (backbone) linkages. The term also includes modified or
substituted sequences comprising non-naturally occurring monomers
or portions thereof. The nucleic acid sequences of the present
application may be deoxyribonucleic acid sequences (DNA) or
ribonucleic acid sequences (RNA) and may include naturally
occurring bases including adenine, guanine, cytosine, thymidine and
uracil. The sequences may also contain modified bases. Examples of
such modified bases include aza and deaza adenine, guanine,
cytosine, thymidine and uracil; and xanthine and hypoxanthine. The
nucleic acids of the present disclosure may be isolated from
biological organisms, formed by laboratory methods of genetic
recombination or obtained by chemical synthesis or other known
protocols for creating nucleic acids.
[0059] The term "isolated polynucleotide" or "isolated nucleic acid
sequence" as used herein refers to a nucleic acid substantially
free of cellular material or culture medium when produced by
recombinant DNA techniques, or chemical precursors, or other
chemicals when chemically synthesized. An isolated nucleic acid is
also substantially free of sequences which naturally flank the
nucleic acid (i.e. sequences located at the 5' and 3' ends of the
nucleic acid) from which the nucleic acid is derived. The term
"nucleic acid" is intended to include DNA and RNA and can be either
double stranded or single stranded, and represents the sense or
antisense strand. Further, the term "nucleic acid" includes the
complementary nucleic acid sequences.
[0060] The term "recombinant nucleic acid" or "engineered nucleic
acid" as used herein refers to a nucleic acid or polynucleotide
that is not found in a biological organism. For example,
recombinant nucleic acids may be formed by laboratory methods of
genetic recombination (such as molecular cloning) to create
sequences that would not otherwise be found in nature. Recombinant
nucleic acids may also be created by chemical synthesis or other
known protocols for creating nucleic acids.
[0061] The term "polypeptide" or "protein" as used herein describes
a chain of amino acids that correspond to those encoded by a
nucleic acid. A polypeptide or protein of this disclosure can be a
peptide, which usually describes a chain of amino acids of from two
to about 30 amino acids. The term protein as used herein also
describes a chain of amino acids having more than 30 amino acids
and can be a fragment or domain of a protein or a full length
protein. Furthermore, as used herein, the term protein can refer to
a linear chain of amino acids or it can refer to a chain of amino
acids that has been processed and folded into a functional protein.
It is understood, however, that 30 is an arbitrary number with
regard to distinguishing peptides and proteins and the terms can be
used interchangeably for a chain of amino acids. The proteins of
the present disclosure can be obtained by isolation and
purification of the proteins from cells where they are produced
naturally, by enzymatic (e.g., proteolytic) cleavage, and/or
recombinantly by expression of nucleic acid encoding the proteins
or fragments of this disclosure. The proteins and/or fragments of
this disclosure can also be obtained by chemical synthesis or other
known protocols for producing proteins and fragments.
[0062] The term "isolated polypeptide" refers to a polypeptide
substantially free of cellular material or culture medium when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized.
[0063] The term "antibody" as used herein is intended to include
monoclonal antibodies, polyclonal antibodies, single chain
antibodies, chimeric antibodies and antibody fusions. The antibody
may be from recombinant sources and/or produced in transgenic
animals. The term "antibody fragment" as used herein is intended to
include without limitations Fab, Fab', F(ab).sub.2, scFv, dsFv,
ds-scFv, dimers, minibodies, diabodies, and multimers thereof,
multispecific antibody fragments and Domain Antibodies.
[0064] The term "vector" as used herein refers to a polynucleotide
that can be used to deliver a nucleic acid to the inside of a cell.
In one embodiment, a vector is an expression vector comprising
expression control sequences (for example, a promoter) operatively
linked to a nucleic acid to be expressed in a cell. Vectors known
in the art include, but are not limited to, plasmids, phages,
cosmids and viruses.
(ii) Compositions
[0065] The present inventors have developed a Trifunctional T
cell-antigen coupler (Tri-TAC) to better mimic the natural
signaling through the T-cell receptor (TCR), while retaining MHC
unrestricted targeting. Specifically, the inventors created a
molecule where the transmembrane and intracellular regions of the
CD4 co-receptor, which localize to the lipid raft and bind Lck,
respectively, were fused to single-chain antibody that binds CD3.
The construct is designed to draw the CD3 molecule and the TCR into
regions of lipid rafts and bring Lck into the proximity of the TCR,
similar to natural MHC binding. To target the chimeric receptor, a
designed ankyrin repeat (DARPin) was linked to the CD4-UCHT1
chimera to generate a tri-functional T cell-antigen coupler
(Tri-TAC).
[0066] Experimentally, human T cells were engineered to express
either the prototype Tri-TAC or a conventional CAR with the same
DARPin. It was determined that in all aspects, T cells engineered
with the Tri-TAC demonstrated functionality equivalent to a
conventional CAR. With regard to two parameters (TNF-.alpha.
production and CD107a mobilization), it was observed that the
Tri-TAC was more active than a conventional CAR. Further, the data
shows that on a per molecule basis the Tri-TAC shows a
significantly enhanced activity. Additionally the Tri-TAC offers
enhanced safety compared to traditional CARs as no activation
domains are part of the protein.
[0067] Accordingly, the present disclosure relates to a nucleic
acid comprising:
[0068] a first polynucleotide encoding a target-specific
ligand;
[0069] a second polynucleotide encoding a ligand that binds the TCR
complex; and
[0070] a third polynucleotide encoding a T cell receptor signaling
domain polypeptide.
[0071] In one embodiment, the nucleic acid is a recombinant, or
engineered, nucleic acid. In another embodiment, the first, second
and/or third polynucleotides are recombinant, or engineered,
polynucleotides.
[0072] The disclosure also relates to a polypeptide encoded by the
nucleic acid and a composition comprising the nucleic acid.
[0073] A nucleic acid comprising each of the first, second and
third polynucleotides, and the polypeptide encoded by the nucleic
acid is also referred to herein as a Trifunctional T cell-antigen
coupler or Tri-TAC.
Target-Specific Ligand
[0074] The target-specific ligand directs the T cell-antigen
coupler to a target cell. Accordingly, a target-specific ligand
refers to any substance that binds, directly or indirectly, to a
target cell. A target cell may be any cell associated with a
disease state, including, but not limited to cancer. In one
embodiment, the target specific ligand binds to an antigen (protein
produced by a cell that can elicit an immune response) on the
target cell. The target-specific ligand can also be referred to as
an antigen binding domain.
[0075] In one embodiment, a target cell is a tumor cell. Here, a
target-specific ligand can bind to a tumor antigen or tumor
associated antigen on a tumor cell. Tumor antigens are well known
in the art. The term "tumor antigen" or "tumor associated antigen"
as used herein means any antigenic substance produced in tumor
cells that triggers an immune response in a host (e.g. which can be
represented by MHC complexes). The tumor antigen when proteinaceous
can for example be a sequence of 8 or more amino acids up to the
full protein and any number of amino acids in between 8 and the
full length protein which comprises at least one antigenic fragment
of the full length protein that can be represented in a MHC
complex. Examples of tumor antigens include, but are not limited
to, HER2 (erbB-2), B-cell maturation antigen (BCMA),
alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125,
MUC-1, epithelial tumor antigen (ETA), tyrosinase,
melanoma-associated antigen (MAGE), prostate-specific antigen
(PSA), glioma-associated antigen, (.beta.-human chorionic
gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase
reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase,
mut hsp70-2, M-CSF, prostase, PAP, NY-ESO-1, LAGE-la, p53,
prostein, PSMA, survivin and telomerase, prostate-carcinoma tumor
antigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin
growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.
[0076] Examples of target-specific ligands include antibodies and
fragments thereof, for example single chain antibodies such as
scFVs, or small proteins that bind to the target cell and/or
antigen.
[0077] One example of a target-specific ligand is a designed
ankyrin [0063] repeat (DARPin) targeted to a specific cell and/or
antigen. In one embodiment, the target-specific ligand is a DARPin
targeted to HER2 (erbB-2). One example of a DARPin targeted to HER2
(erb-2) is provided herein as SEQ ID NO: 7 and 8.
[0078] Another example of a target-specific ligand is a scFV
targeted to [0064] a specific cell and/or antigen. In one
embodiment, the target-specific ligand is a scFv that binds HER2
(erb-2). One example of an scFv that binds HER2 (erb-2) is provided
herein as SEQ ID NO: 22 and 23.
Ligand that Binds the TCR Complex
[0079] The T cell-antigen coupler is designed to recruit the T-Cell
Receptor (TCR) in combination with co-receptor stimulation.
Accordingly, the T cell antigen coupler includes a ligand that
binds a protein associated with the T-cell receptor complex.
[0080] The TCR (T-Cell Receptor) is a complex of integral membrane
proteins that participates in the activation of T cells in response
to the binding of an antigen. The TCR is a disulfide-linked
membrane-anchored heterodimer normally consisting of the highly
variable alpha (.alpha.) and beta (.beta.) chains expressed as part
of a complex with the invariant CD3 (cluster of differentiation 3)
chain molecules. T cells expressing this receptor are referred to
as .alpha.:.beta. (or .alpha..beta. T cells, though a minority of T
cells express an alternate receptor, formed by variable gamma
(.gamma.) and delta (.delta.) chains, referred as .gamma..delta. T
cells. CD3 is a protein complex composed of four distinct chains.
In mammals, the complex contains a CD3.gamma. chain, a CD3.delta.
chain, and two CD3.epsilon. chains.
[0081] As used herein, the term "ligand that binds a protein
associated with the T-cell receptor complex" includes any substance
that binds, directly or indirectly, to a protein of the TCR.
Proteins associated with the TCR include, but are not limited to
the TCR alpha (.alpha.) chain, TCR beta (.beta.) chain, TCR gamma
(.gamma.) chain, TCR delta (.delta.) chain, CD3.gamma. chain,
CD3.delta. chain and CD3.epsilon. chains. In one embodiment, a
ligand that binds a protein associated with the T-cell receptor
complex is an antibody to the TCR alpha (.alpha.) chain, TCR beta
(.beta.) chain, TCR gamma (.gamma.) chain, TCR delta (.delta.)
chain, CD3.gamma. chain, CD3.delta. chain and/or CD3.epsilon.
chain.
[0082] In one embodiment, the ligand is an antibody or a fragment
thereof that binds CD3. Examples of CD3 antibodies are known in the
art (for muromonab, otelixizumab, teplizumab and visilizumab). In
one embodiment, the antibody that binds CD3 is a single chain
antibody, for example a singlechain variable fragment (scFv).
[0083] Another example of a CD3 antibody is UCHT1 which targets
CD3c. A sequence for UCHT1 is provided herein as SEQ ID NOs: 13 and
14.
T Cell Receptor Signaling Domain Polypeptide
[0084] The T cell antigen coupler includes a T cell receptor
signaling domain polypeptide. As used herein, the term "T cell
receptor signaling domain" refers to a polypeptide that (a)
localizes to the lipid raft and/or (b) binds Lck. A T cell receptor
signaling domain polypeptide can include one or more protein
domains including, but not limited to, a cytoplasmic domain and/or
a transmembrane domain. As used herein, "protein domain" refers to
a conserved part of a given protein sequence structure that can
function and exist independently of the rest of the protein chain.
In one embodiment, the T cell receptor signaling domain polypeptide
includes a cytoplasmic domain. In another embodiment, the T cell
receptor signaling domain polypeptide includes a transmembrane
domain. In a further embodiment, the T cell receptor signaling
domain polypeptide includes both a cytoplasmic and a transmembrane
domain.
[0085] T cell receptor signaling domain polypeptides include TCR
co-receptors and co-stimulators and TCR co-receptor and
co-stimulator protein domains.
[0086] A "TCR co-receptor" refers to a molecule that assists the T
cell receptor (TCR) in communicating with an antigen-presenting
cell. Examples of TCR co-receptors include, but are not limited to,
CD4, CD8, CD28, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37,
CD64, CD80, CD86, CD134, CDt 37 and CD 154.
[0087] A "TCR co-stimulator" refers to a molecule that is required
for the response of a T cell to an antigen. Examples of TCR
co-stimulators include, but are not limited to, PD-1, ICOS, CD27,
CD28, 4-1BB (CD 137), OX40, CD30, 25 CD40, lymphocyte
fiction-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C,
B7-H3, and a ligand that specifically binds CD83.
[0088] In one embodiment, the T cell receptor signaling domain
polypeptide includes both a cytoplasmic domain and a transmembrane
domain of a TCR co-receptor or co-stimulator protein. The
cytoplasmic domain and transmembrane domain may be from the same
co-receptor or co-stimulator or from different co-receptors or
co-stimulators. The cytoplasmic domain and transmembrane domains
are optionally joined by a linker.
[0089] In one embodiment, the T cell receptor signaling domain
polypeptide comprises the transmembrane and cytoplasmic domains of
the CD4 co-receptor (see for example SEQ ID NO: 17 and 18).
[0090] In another embodiment, the T cell receptor signaling domain
polypeptide comprises the transmembrane and cytoplasmic domains of
the CD8a co-receptor.
[0091] In other embodiment, the cytoplasmic and/or transmembrane
domain of the T cell receptor signaling domain polypeptide is
synthetic. For example, the transmembrane domain is optionally a
synthetic, highly hydrophobic membrane domain.
[0092] In another example, the transmembrane domain is a
glycophorine transmembrane domain. In yet another example the T
cell receptor signaling domain polypeptide includes a CD48 GPI
signal sequence to attach the T-cell antigen coupler to the
membrane using the GPI anchor.
[0093] In addition to the three components of the T cell antigen
coupler described herein (target-specific ligand, ligand that binds
the TCR complex and T cell receptor signaling domain polypeptide),
it is contemplated that other polypeptides could also be included.
For example, the T cell antigen coupler optionally includes
additional polypeptides that directly or indirectly act to target
or activate the T cell.
Linkers
[0094] The various components of the T cell antigen coupler can be
fused directly to each other, or they may be joined by at least one
linker, optionally a peptide linker. The peptide linker can be of
any size provided it does not interfere with the function of the
individual linked components. In one embodiment, the peptide linker
is from about 1 to about 15 amino acids in length, more
specifically from about 1 to about 10 amino acids, and most
specifically from about 1 to about 6 amino acids.
[0095] Examples of linkers useful in the T cell antigen coupler
include the G453 linker. Other examples of linkers are peptides
corresponding to SEQ ID NOs: 11, 12, 15, 16, 19, 20 and 21 and
variants and fragments thereof:
Configuration
[0096] The T cell-antigen coupler may be present in various
configurations as will be readily appreciated by a person of skill
in the art.
[0097] In one embodiment, the target specific ligand and the T cell
receptor signaling domain polypeptide are both fused to the ligand
that binds the TCR complex. For example, the N-DARPin TAG described
here (also referred to as configuration 1; SEQ ID NO: 1 and 2)
includes, in order: [0098] i) N-Darpin Tri TAG leader sequence
(secretion signal) (SEQ ID NO: 5 and 6) [0099] ii) DARPin specific
for Her2 antigen (SEQ ID NO: 7 and 8) [0100] iii) Myc tag (SEQ ID
NO: 9 and 10) [0101] iv) Linker 1 (SEQ ID NO: 11 and 12) [0102] v)
UCHT1 (SEQ ID NO: 13 and 14) [0103] vi) Linker 2 (SEQ ID NO: 15 and
16) [0104] vii) CD4 (SEQ ID NO: 17 and 18)
[0105] In another embodiment, the DARPin is replaced with a scFV
ScFv specific for a Her2 antigen (SEQ ID NO: 22 and 23).
[0106] In another embodiment, the ligand that binds the TCR complex
and the T cell receptor signaling domain polypeptide are both fused
to the target specific ligand (C-DARPin TAG as described here (also
referred to as configuration 1; SEQ ID NO: 3 and 4)). Alternative
configurations will be readily apparent to a person of skill in the
art.
Vector Constructs
[0107] A variety of delivery vectors and expression vehicles can be
employed to introduce nucleic acids described herein into a cell.
Accordingly, the aforementioned polynucleotides are optionally
comprised in a vector to provide a vector construct, also herein
referred to as a vector.
[0108] Therefore, the present disclosure also relates to a vector
comprising:
[0109] a. a first polynucleotide encoding a target-specific
ligand;
[0110] b. a second polynucleotide encoding an antibody that binds
CD3; and
[0111] c. a third polynucleotide encoding a T cell receptor
signaling domain polypeptide, and optionally a promoter functional
in a mammalian cell.
[0112] Promoters, regions of DNA that initiate transcription of a
particular nucleic acid sequence, are well known in the art. A
"promoter functional in a mammalian cell" refers to a promoter that
drives expression of the associated nucleic acid sequence in a
mammalian cell. A promoter that drives expression of a nucleic acid
sequence may be referred to as being "operably connected" to the
nucleic acid sequence.
[0113] In one embodiment, the first polynucleotide and third
polynucleotide are fused to the second polynucleotide to provide a
T cell antigen coupler fusion and the coding sequence of the T cell
antigen coupler fusion is operably connected to the promoter.
[0114] In another embodiment, the second polynucleotide and third
polynucleotide are fused to the first polynucleotide to provide a T
cell antigen coupler fusion and the coding sequence of the T cell
antigen coupler fusion is operably connected to the promoter.
[0115] Optionally, the vector is designed for expression in
mammalian cells such as T cells. In one embodiment, the vector is a
viral vector, optionally a retroviral vector.
[0116] Vectors that are useful comprise vectors derived from
lentiviruses, Murine Stem Cell Viruses (MSCV), pox viruses,
oncoretroviruses, adenoviruses, and adeno-associated viruses. Other
delivery vectors that are useful comprise vectors derived from
herpes simplex viruses, transposons, vaccinia viruses, human
papilloma virus, Simian immunodeficiency viruses, HTLV, human foamy
virus and variants thereof. Further vectors that are useful
comprise vectors derived from spumaviruses, mammalian type B
retroviruses, mammalian type C retroviruses, avian type C
retroviruses, mammalian type D retroviruses and HTLV/BLV type
retroviruses. One example of a lentiviral vector useful in the
disclosed compositions and methods is the pCCL vector.
Variations of Polynucleotides and Polypeptides
[0117] Many modifications may be made to the polynucleotide
sequences including vector sequences and polypeptides sequences
disclosed in this application and these will be apparent to one
skilled in the art. Modifications include substitution, insertion
or deletion of nucleotides or amino acids or altering the relative
positions or order of nucleotides or amino acids.
[0118] In one embodiment, the polynucleotides described herein may
be modified or mutated to optimize the function of the encoded
polypeptide and/or the function, activity and/or expression of the
T cell antigen coupler.
[0119] It is shown herein that UCHT1 mutant can be generated that
results in enhanced surface expression of the TAG (FIGS. 15-17).
Accordingly, in one embodiment, the TAG comprises a modified or
mutated ligand that binds the TCR complex, wherein the TAG
comprising the modified or mutated antibody has increased surface
expression and/or activity compared to a TAG comprising a wild
type, or non-modified or mutated ligand that binds the TCR complex.
An example of a mutated or modified antibody that binds CD3 is the
UCHT1 A85V, T161P mutant described herein (SEQ ID NO: 24 and
25).
Sequence Identity
[0120] The polynucleotides of the application also include nucleic
acid molecules (or a fragment thereof) having at least about: 70%
identity, at least 80% identity, at least 90% identity, at least
95% identity, at least 96% identity, at least 97% identity, at
least 98% identity or, at least 99% or 99.5% identity to a nucleic
acid molecule of the application. The polypeptides of the
application also include polypeptides (or a fragment thereof)
having at least about: 70% identity, at least 80% identity, at
least 90% identity, at least 95% identity, at least 96% identity,
at least 97% identity, at least 98% identity or, at least 99% or
99.5% identity to a polypeptide of the application. Identity refers
to the similarity of two nucleotide or polypeptide sequences that
are aligned so that the highest order match is obtained. Identity
is calculated according to methods known in the art. For example,
if a nucleotide sequence (called "Sequence A") has 90% identity to
a portion of SEQ ID NO: 1, then Sequence A will be identical to the
referenced portion of SEQ ID NO: 1 except that Sequence A may
include up to 10 point mutations (such as substitutions with other
nucleotides) per each 100 nucleotides of the referenced portion of
SEQ ID NO:
[0121] Sequence identity is preferably set at least about: 70%
identity, at least 80% identity, at least 90% identity, at least
95% identity, at least 96% identity, at least 97% identity, at
least 98% identity or, most preferred, at least 99% or 99.5%
identity to the nucleotide sequences provided herein and/or its
complementary sequence. Sequence identity is also preferably set at
least about: 70% identity, at least 80% identity, at least 90%
identity, at least 95% identity, at least 96% identity, at least
97% identity, at least 98% identity or, most preferred, at least
99% or 99.5% identity to the polypeptide sequences provided herein.
Sequence identity will preferably be calculated with the GCG
program from Bioinformatics (University of Wisconsin). Other
programs are also available to calculate sequence identity, such as
the Clustal W program (preferably using default parameters;
Thompson, J D et al., Nucleic Acid Res. 22:4673-4680).
Hybridization
[0122] The application includes DNA that has a sequence with
sufficient identity to a nucleic acid molecule described in this
application to hybridize under stringent hybridization conditions
(hybridization techniques are well known in the art). The present
application also includes nucleic acid molecules that hybridize to
one or more of the sequences described herein and/or its
complementary sequence. Such nucleic acid molecules preferably
hybridize under high stringency conditions (see Sambrook et al.
Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). High
stringency washes have preferably have low salt (preferably about
0.2% SSC) and a temperature of about 50-65.degree. C. and are
optionally conducted for about 15 minutes.
Expression in T Cells
[0123] The T cell antigen coupler is designed for expression in T
cells. Accordingly, one aspect of the disclosure provides a T cell
expressing a T cell antigen coupler. Another aspect of the
disclosure relates to a T cell transduced or transfected with T
cell antigen coupler or a vector comprising a T cell antigen
coupler. Optionally, the T cell is an isolated T cell.
[0124] T cells can be obtained from a number of sources, including,
but not limited to blood (for example, peripheral blood mononuclear
cells), bone marrow, thymus tissue, lymph node tissue, cord blood,
thymus tissue, tissue from an infection site, spleen tissue, and
tumors. In one embodiment, the T cells are autologous T cells. In
another embodiment, the T cells are obtained from a cell line of T
cells. Methods of culturing and maintaining T cells in vitro are
well known in the art.
[0125] Once obtained, the T cells are optionally enriched in vitro.
As is well known in the art, a population of cells can be enriched
by positive or negative selection. Further, the T cells can be
optionally frozen or cryopreserved and then thawed at a later
date.
[0126] Before or after introducing the T cell antigen coupler to
the T cells, the T cells are optionally activated and/or expanded
using methods well known in the art. For example, the T cells can
be expanded by contact with a surface having attached thereto an
agent that stimulates a CD3/TCR complex associated signal and a
ligand that stimulates a co-stimulator molecule on the surface of
the T cells.
[0127] Methods of transducing or transfecting T cells with nucleic
acid sequences and expressing the transduced nucleic acids in the T
cells are well known in the art. For example, a nucleic acid can be
introduced into a cell by physical, chemical or biological means.
Physical means include, but are not limited to, (microinjection,
electroporation, particle bombardment, lipofection and calcium
phosphate precipitation). Biological means include the use of DNA
and RNA vectors.
[0128] In one embodiment, viral vectors, including retroviral
vectors, are used to introduce and express a nucleic acid into a T
cell. Viral vectors include vectors derived from lentivirus, Murine
Stem Cell Viruses (MSCV), pox viruses, herpes simplex virus I
adenovirus and adeno-associated viruses. The vector optionally
includes a promoter that drives expression of the transduced
nucleic acid molecule in a T cell.
[0129] Various assays may be used to confirm the presence and/or
expression of the transduced nucleic acid sequence and/or the
polypeptide encoded by the nucleic acid in the T cell. Assays
include, but are not limited to Southern and Northern blotting,
RT-PCR and PCR, ELISAs and Western blotting.
[0130] In one embodiment, a T cell expressing a T cell antigen
coupler has increased T cell activation in the presence of an
antigen compared to a T cell not expressing a T cell antigen
coupler and/or as compared to a T cell expressing a traditional
CAR. Increased T cell activation can be ascertained by numerous
methods, including but not limited to, increased tumor cell line
killing, increased cytokine production, increased cytolysis,
increased degranulation and/or increased expression of activation
markers such as CD107.alpha., IFN.gamma., 11-2 or TNF.alpha..
Increases may be measured in an individual cell or in a population
of cells.
[0131] The terms "increased" or "increasing" as used herein refer
to at least a 2%, 5%, 10%, 25%, 50%, 100% or 200% increase in a T
cell or population of T cells expressing a T cell antigen coupler
compared to a T cell or population of T cells not expressing a T
cell antigen coupler and/or as compared to a T cell or population
of T cells expressing a traditional CAR.
[0132] T cells, optionally autologous T cells, expressing the T
cell antigen coupler can be administered to a subject in need
thereof. According, a T cell transduced with and/or expressing a T
cell antigen coupler can be formulated in a pharmaceutical
composition. Preferably, the T cells are formulated for intravenous
administration.
[0133] A pharmaceutical composition can be prepared by per se known
methods for the preparation of pharmaceutically acceptable
compositions that can be administered to subjects, such that an
effective quantity of the T cells are combined in a mixture with a
pharmaceutically acceptable carrier. Suitable carriers are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing
Company, Easton, Pa., USA, 2000). On this basis, the compositions
include, albeit not exclusively, solutions of the substances in
association with one or more pharmaceutically acceptable carriers
or diluents, and contained in buffered solutions with a suitable pH
and iso-osmotic with the physiological fluids.
[0134] Suitable pharmaceutically acceptable carriers include
essentially chemically inert and nontoxic compositions that do not
interfere with the effectiveness of the biological activity of the
pharmaceutical composition. Examples of suitable pharmaceutical
carriers include, but are not limited to, water, saline solutions,
glycerol solutions, ethanol,
N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride
(DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
Such compositions should contain a therapeutically effective amount
of the compound, together with a suitable amount of carrier so as
to provide the form for direct administration to the patient.
[0135] Pharmaceutical compositions may also include, without
limitation, lyophilized powders or aqueous or non-aqueous sterile
injectable solutions or suspensions, which may further contain
antioxidants, buffers, bacteriostats and solutes that render the
compositions substantially compatible with the tissues or the blood
of an intended recipient. Other components that may be present in
such compositions include water, surfactants (such as Tween),
alcohols, polyols, glycerin and vegetable oils, for example.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules, tablets, or concentrated solutions
or suspensions.
(iii) Methods and Uses
[0136] One aspect of the present disclosure provides the use of a
Trifunctional T-cell antigen coupler to direct a T cell to a
specific antigen.
[0137] Accordingly, the present disclosure also relates to the use
of a modified T cell for treating cancer in a subject in need
thereof, wherein the modified T cell expresses a nucleic acid
comprising a first polynucleotide encoding a target-specific
ligand, a second polynucleotide encoding an ligand that binds the
TCR complex; and a third polynucleotide encoding a T cell receptor
signaling domain polypeptide. The disclosure also relates to
methods for treating cancer, comprising administering an effective
amount of modified T cells to a subject in need thereof. Also
disclosed is use of an effective amount of modified T cells for
treating cancer in a subject in need thereof. Further disclosed is
use of a modified T cell in the preparation of a medicament
treating cancer in a subject in need thereof. Even further
disclosed is a modified T cell for use in treating cancer in a
subject in need thereof. In one embodiment, the target-specific
ligand binds an antigen on a cancerous cell, thereby targeting the
modified T cell to the cancerous cell.
[0138] Cancers that may be treated include any form of neoplastic
disease. Examples of cancers that may be treated include, but are
not limited to breast cancer, lung cancer and leukemia, for example
mixed lineage leukemia (MLL), chronic lymphocytic leukemia (CLL) or
acute lymphoblastic leukemia (ALL). Other cancers include
carcinomas, blastomas, melonomas, sarcomas, hematological cancers,
lymphoid malignancies, benign and malignant tumors, and
malignancies. The cancer can comprise non-solid tumors or solid
tumors. Cancers that may be treated include tumors that are not
vascularized, or not yet substantially vascularized, as well as
vascularized tumors.
[0139] The modified T cells and/or pharmaceutical compositions
described herein may be administered to, or used in, living
organisms including humans, and animals. The term "subject" as used
herein refers to any member of the animal kingdom, preferably a
mammal, more preferably a human being.
[0140] Procedures for isolating, genetically modifying, and
administering T cells to a subject in need thereof are known in the
art. In particular, T cells are isolated from a mammal (preferably
a human), optionally expanded and/or activated as described herein
and transduced or transfected with the nucleic acid molecules of
the disclosure. The T cells can be autologous with respect to the
subject. In another embodiment, the cells can be allogeneic,
syngeneic or xenogeneic with respect to the subject.
[0141] The modified T cells can be administered either alone, or as
a pharmaceutical composition, as described herein. Compositions of
the present disclosure are preferably formulated for intravenous
administration.
[0142] Administration of an "effective amount" of the modified T
cells and/or pharmaceutical compositions is defined as an amount
effective, at dosages and' for periods of time necessary to achieve
the desired result. For example, an effective amount of a substance
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the recombinant
protein to elicit a desired response in the individual. Dosage
regime may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the
dose may be proportionally reduced as indicated by the exigencies
of the therapeutic situation.
[0143] For example, the modified T cells and/or pharmaceutical
compositions described herein may be administered at a dosage of
10.sup.4 to 10.sup.9 cells per kg body weight, optionally 10.sup.5
to 10.sup.8 cells per kg body weight or 10.sup.6 to 10.sup.7 cells
per kg body weight. The dosage can be administered a single time or
multiple times.
[0144] The modified T cells and/or pharmaceutical compositions may
be administered by any method known in the art, including but not
limited to, aerosol inhalation, injection, ingestion, transfusion,
implantation or transplantation. The modified T cells and/or
pharmaceutical compositions may administered to a subject
subcutaneously, intradennally, intratumorally, intranodally,
intrameduliary, intramuscularly, by intravenous (i.v.) injection,
or intraperitoneally. The modified T cells and/or pharmaceutical
compositions thereof may be injected directly into a tumor, lymph
node, or site of infection.
[0145] As used herein, and as well understood in the art, "to
treat" or "treatment" is an approach for obtaining beneficial or
desired results, including clinical results. Beneficial or desired
clinical results can include, but are not limited to, alleviation
or amelioration of one or more symptoms or conditions, diminishment
of extent of disease, stabilized (i.e. not worsening) state of
disease, preventing spread 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. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. In one
embodiment, "treatment" includes preventing a disease or
condition.
TABLE-US-00001 SEQ ID NO Description Nucleotide/Amino Acid SEQ ID
NO: 1 N-Darpin Tri TAC Nucleotide SEQ ID NO: 2 N-Darpin Tri TAC
Amino Acid SEQ ID NO: 3 C-Darpin Tri TAC Nucleotide SEQ ID NO: 4
C-Darpin Tri TAC Amino Acid SEQ ID NO: 5 N-Darpin Tri TAC
Nucleotide leader sequence (secretion signal) SEQ ID NO: 6 N-Darpin
Tri TAC Amino Acid leader sequence (secretion signal) SEQ ID NO: 7
DARPin specific Nucleotide for Her2 antigen SEQ ID NO: 8 DARPin
specific Amino Acid for Her2 antigen SEQ ID NO: 9 Myc Tag
Nucleotide SEQ ID NO: 10 Myc Tag Amino Acid SEQ ID NO: 11 Linker 1
Nucleotide SEQ ID NO: 12 Linker 1 Amino Acid SEQ ID NO: 13
UCHT1.sup.1 Nucleotide SEQ ID NO: 14 UCHT1.sup.2 Amino Acid SEQ ID
NO: 15 Linker 2 Nucleotide SEQ ID NO: 16 Linker 2 Amino Acid SEQ ID
NO: 17 CD4 Domain.sup.3 Nucleotide SEQ ID NO: 18 CD4 Domain.sup.4
Amino Acid SEQ ID NO: 19 Universal rigid Amino Acid linker SEQ ID
NO: 20 CD28 based linker Amino Acid SEQ ID NO: 21 CD4 based linker
Amino Acid SEQ ID NO: 22 ScFv specific for Nucleotide Her2 antigen
SEQ ID NO: 23 ScFv specific for Amino Acid Her2 antigen SEQ ID NO:
24 UCHT1 (A85V, T161P) Nucleotide SEQ ID NO: 25 UCHT1 (A85V, T161P)
Amino Acid .sup.1Light chain, nucleotides 1-324; Linker,
nucleotides 325-387; Heavy chain, nucleotides 388-750 .sup.2Light
chain, amino acids 1-108; Linker, amino acids 109-128; Heavy chain,
amino acids 129-250 .sup.3Extracellular linker, nucleotides 1-66;
Transmembrane domain, nucleotides 67-132; Cytosolic domain,
nucleotides 133-254 .sup.4Extracellular linker, amino acids 1-22;
Transmembrane domain, amino acids 23-44; Cytosolic domain, amino
acids 45-84
[0146] The above disclosure generally describes the present
application. A more complete understanding can be obtained by
reference to the following specific examples. These examples are
described solely for the purpose of illustration and are not
intended to limit the scope of the application. Changes in form and
substitution of equivalents are contemplated as circumstances might
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitation.
[0147] The following non-limiting examples are illustrative of the
present application.
EXAMPLES
Example 1
Background and Summary
[0148] A trifunctional T cell-antigen coupler (Tri-TAC) was
developed to better recapitulate the natural signaling through the
TCR, while retaining MHC unrestricted targeting. T-cell activation
occurs following ligation of MHC by the TCR and co-receptor on the
T cells (either CD4 or CD8) simultaneously bind to conserved
regions within the MHC molecule (Yin et al., 2012)(Kuhns and Davis,
2012). The co-receptors are specifically located within "lipid
rafts" (Fragoso et al., 2003XArcaro et al., 2000), membrane micro
domains that are particularly important for TCR signal complex
formation (He and Marguet, 2008). In addition to ensuring the
correct microdomain localization of the TCR activation complex,
these co-receptors also bind directly to Lck (Kim et al., 2003), a
protein kinase that is crucial for T-cell activation (Methi et al.,
2005)(Acuto and Cantrell, 2000). As stated previously, none of the
existing chimeric receptors or bi-functional proteins engage the
co-receptor molecules or Lck. A molecule was created where the
transmembrane and intracellular regions of the CD4 co-receptor,
which localize to the lipid raft and bind Lck, respectively, were
fused to single-chain antibody that binds CD3 (UCHT1; SEQ ID NO: 13
and 14; sequence is also in the public domain). This construct is
designed to draw the CD3 molecule and the TCR into regions of lipid
rafts and bring Lck into the proximity of the TCR, similar to
natural MEW binding. To target this chimeric receptor, a designed
ankyrin repeat (DARPin) was linked to the CD4-UCHT1 chimera to
generate a tri-functional T cell-antigen coupler (Tri-TAC). In this
specific case, the DARPin was specific for the proto-oncogene,
erbB-2.
[0149] Human T cells were engineered to express either the
prototype Tri-TAC or a conventional CAR with the same DARPin. It
was determined that in all aspects, T cells engineered with the
Tri-TAC demonstrated functionality equivalent to a conventional
CAR. Interestingly, with regard to 2 parameters (TNF-.alpha.
production and CD107a mobilization), it was observed that the
Tri-TAC was more active than a conventional CAR. Further, the data
shows that on a per molecule basis the Tri-TAC shows a
significantly enhanced activity. Additionally the Tri-TAC offers
enhanced safety compared to traditional CARs as no activation
domains are part of the protein.
[0150] The traditional CAR is effective in stimulating T-cells by
combining several signaling domains (FIG. 1C). By comparison, the
Tri-TAC (FIG. 1 A/B) does not contain any signaling domains of its
own. It relies purely on facilitating the proposed interactions
between other key players (shown in grey) in an antigen dependent
manner. To test this design hypothesis, several variants of the
full length N-Darpin Tri-TAC were generated (FIG. 1D).
[0151] Previous work has established the consistent and significant
cell surface expression of CAR molecules. It was found that the
Darpin CAR shows robust surface expression (FIG. 2). In contrast,
Tri-TAC showed a much lower surface expression. This was observed
for all variants that had the UCHT1 domain. However, the Tri-TAC
variant lacking the UCHT1 domain showed surface expression similar
to the Darpin CAR.
[0152] T cells engineered to express the Tri-TAC, the Tri-TAC
variants or The T cells the DARPin CAR were stimulated with
plate-bound antigen, engineered to express the Tri-TAC and DARPin
CAR could elaborate all measured functions (TNF-.alpha. production,
IFN-.gamma. production and CD107a mobilization) (FIGS. 3A and 3B).
Binding of the Tri-TAC to both CD3 and the target antigen was found
to be critical for the T cells to elaborate their functions. In
FIG. 3, it is demonstrated that removal of UCHT1, which abolishes
binding to CD3, abrogates the function of the Tri-TAC. In other
data, it was determined that removal of the DARPin from the Tri-TAC
also abrogates function.
[0153] As expected, when these T-cells were tested for their
cytotoxicity, Tri-TAC-UCHT1-Darpin showed no ability to kill
antigen expressing cells (FIG. 4). N-Darpin Tri-TAC showed a high
level of selective cytotoxicity that was very similar to the
classical DARPin-CAR. Interestingly, the T cells expressing the
DARPin-CAR appear to display off-target killing at high
T-cell:target cell ratios (see killing on D2F2 in FIG. 4) whereas
the T cells expressing the Tri-TAC did not display these
effects.
Experimental
[0154] FIG. 1 is a schematic overview. (A) Depicts N-Darpin
Tri-TAC. The ankyrin repeat domain targeted against Her2 is fused
to the single chain fragment variable (scFv) UCHT1 using a
(G.sub.4S).sub.3 linker. The scFv is then linked to the CD4
molecule. The CD4 contains the linker region and transmembrane
region as well as the cytoplasmic anchoring region. Potential
interactions are shown in faded gray. (B) Depicts the C-Darpin
Tri-TAC. In this construct, the scFv UCHT1 is switched with the
Darpin domain. Potential interactions are again depicted in faded
gray. (C) Model of a classical second generation CAR. The Darpin
targeting domain is linked via a CD8a linker to the CD28
transmembrane domain. The CD3 zeta domain with its 3 activating
ITAM motifs is then connected to the cytosolic portion of CD28. (D)
Overview of the various Tri-TAC controls, lacking either the Darpin
targeting domain, the CD3 binding scFv moiety, or the cytosolic
portion of the CD4 domain.
[0155] FIG. 2 shows the phenotypic surface expression analysis of
transduced T-cells with histograms of respective Tri-TAC variants.
T-cells had been incubated with Her2Fc, which later was detected
via flow cytometry. The presented data was gated on CD8+
lymphocytes. The shown gates were chosen based on the untransduced
control.
[0156] FIG. 3 is a functional analysis of engineered T-cells. In
(A), cells were stimulated for 4 hours with plate bound Her2Fc in
media containing GolgiPlug.TM.. Cells were first stained for CD8+,
then permeabilized and analyzed for TNF-.alpha. and IFN-.gamma.
production. Initial gates were set for singlet CD8+ lymphocytes.
The shown gates were set based on the untransduced control. In B),
as before, cells were stimulated with plate bound Her2Fc. Media
included GolgiPlug.TM. as well as an anti-CD107a antibody. Actively
degranulating cells were expected to have a higher rate of CD107a
recycling, and subsequently show a higher signal for
anti-CD107a.
[0157] FIG. 4 shows engineered T-cell cytotoxicity. Two different
adherent mouse tumor lines were plated 24 hours prior to T-cell
addition. D2F2/E2 have been engineered to express human Her2,
whereas the D2F2 do not. Indicated ratios of T-cells were added to
tumor containing wells. Tumor cells were incubated for 6h with
T-cells. T-cells were subsequently removed via washing. 10% Alamar
blue containing media was added to each well for 3 hours. The
metabolic activity, as an indicator of cell survival, was
determined via endpoint analysis. Wells without T-cells were
defined as maximum survivability/metabolic activity and set to
100%, whereas media incubated without cells was set as 0% metabolic
activity. Data presented is the average of 3 replicates.
Discussion
[0158] Using chimeric receptors to redirect T-cells towards
specific targets in an MHC-independent manner is an attractive
method to treat cancer and may be applicable to infectious diseases
where antigens from the pathogen are found on the plasma membrane.
The chimeric receptor would result in: (1) specific cytotoxicity
against the target cells and (2) minimal off target toxicity.
Conventional CARs are limited in this regard because they rely upon
a synthetic structure where signaling domains are located in
unnatural positions where they may not receive proper regulation
and, thus, there is reduced cellular control of specific
activity.
[0159] The Tri-TAC was designed to re-direct the signaling
components of the natural TCR without employing ectopic
localization of signaling domains. The Tri-TAC was designed with
the following principles: (1) the chimeric receptor should interact
and facilitate ordered assembly of key activating protein
complexes, (2) the chimeric receptor should take advantage of
preexisting cellular adaptations, such as micro-domain environments
and (3) the chimeric receptor should not possess any activating
domains. The Tri-TAC is able to achieve this efficiently and, as
the data demonstrates, at rates of activation that are equal to, if
not better than, that of a 2nd generation CAR.
[0160] This Tri-TAC is thus ideally suited for further integration
with additional designed co-receptors to further fine tune T-cell
activation. Ultimately this should lead to much reduced off target
effects without compromising on targeted cytotoxicity. Tri-TAC
appears to exhibit lower toxicity than existing CARs. Darpin CARs
show mild off target killing at high cell to target ratios, which
may become problematic when used in therapies. However, Tri-TAC,
which is as functional as the traditional CAR, did not display
off-target effects. Since DARPins bind targets with high affinity,
off-target effects may be more common on cells that express high
levels of a chimeric receptor that employs a DARPin. Therefore,
without being bound by theory, the low surface expression of the
Tri-TAC may be advantageous as it reduces the likelihood of such
off-target effects.
[0161] Ultimately, the modular nature of the Tri-TAC technology
allows much more sophisticated fine tuning of the T-cell activation
process. For example, the recruitment of the TCR complex could be
modulated by engineering Tri-TAC molecules with a lower CD3
affinity. This could be used to mimic the natural low TCR affinity
(Chervin et al., 2009) while retaining a high affinity targeting
domain to detect cancer targets. Unlike the classical CAR, the
Tri-TAC technology can be engineered to more closely resemble
this.
[0162] In conclusion, the presented Tri-TAC technology is a highly
efficient molecular tool that is able to (1) efficiently trigger
T-cell activation and cytotoxicity, (2) is able to do this by
mimicking natural T-cell activation and (3) does not require
activation domains of its own.
Example 2. Characterization of the Tri-TAC Technology
[0163] An overview of the Tri-TAC technology is provided in FIG.
5.
[0164] FIG. 5A shows an example of CD8 T-cell activation based on
the co-assembly of different receptors and their associated protein
partners. Initially, the major histocompatibility complex I is
presenting an antigen (helix). This is recognized by a T cell
receptor (TCR) complex capable of binding the antigen. The TCR
complex contains several individual subunits. The .alpha./.beta.
domains are able to interact directly with the antigen presented on
MHC-I. The .alpha./.beta. domains then interact with several other
domains (.epsilon., .gamma., .delta., and .zeta., all of which
participate in T-cell activation via various intracellular
activation domains. The TCR complex interacts with MHC-I
concurrently with the CD8 co-receptor. The CD8 co-receptor binds to
the MHC-I in an antigen independent manner. CD8 directly interacts
with Lck, a protein kinase important for activating the TCR
receptor complex. The CD8 and Lck interaction also ensures their
association with lipid rafts (membrane portion) microdomains, which
are hypothesised to organize and encapsulate other relevant
signalling moieties (dark spheres). Later stages of activation then
lead to CD28 recruitment. If this interaction cascade occurs
several times in parallel, T-cells become activated and are able to
exert their cytotoxic effects.
[0165] FIG. 5B provides an overview of Chimeric Antigen Receptors
(CAR). CARs seek to reproduce the complex mechanism of T-cell
activation by combining several key activation domains, such as
.zeta. and CD28, in a single synthetically engineered molecule. The
CAR then directly interacts with an antigen of choice using
specific binding domains. Depicted here is an ankyrin repeat
protein (DARPin). It is believed that several such interactions
occurring in parallel lead to T-cell activation.
[0166] FIG. 5C depicts bispecific T-cell engager (BiTE) like
molecules which engage T-cells by directly cross linking the TCR
complex to an antigen of choice. The BiTE like molecule depicted
here contains two binding domains. The DARPin moiety is interacts
with the target antigen. The single chain variable fragment domain
(scFv) binds the TCR complex via its epsilon domain. Several such
crosslinkings occurring in parallel lead to T-cell activation.
[0167] FIG. 5D is an overview of the TAC technology mimicking the
natural activation process. The T-cell Antigen Coupler (TAC) is
able to bind to its antigen via the DARPin binding domain. DARPin
is then linked to a scFv able to bind the epsilon domain of the TCR
complex. The TAC then associates with the CD4 transmembrane and
cytosolic domain. CD4, like CD8, interacts with Lck and is situated
in lipid rafts. Thus, TACs combine TCR recruitment with co-receptor
stimulation. Without being bound by theory, it is believed that
several such interactions happening in parallel lead to T-cell
activation.
[0168] Different configurations of the TAC molecule are possible.
FIG. 6A shows a model of the TAC molecule in configuration 1. The
CD4-tail, 5 transmembrane, and linker domains are combined with the
TCR-epsilon specific scFv (UCHT1). The scFv is then linked to the
antigen binding domain. This domain is exchangeable. In this
proposal the antigen binding domains used are either a scFv or
DARPin domain specific for the Her2 antigen. FIG. 6B shows a TAC
molecule in configuration 2. Here, the CD4 domains first interact
with the antigen binding domain. This domain is then linked to the
TCR recruiting scFv (UCHT1) domain.
[0169] FIG. 7 shows the functionality of scFv CD4 TAC. FIG. 7A is a
histogram showing surface expression of the scFv CD4 TAC receptor
relative to empty vector. Cells were stained using an FcHer2
antigen, which was in turn detected using fluorescently labelled
antibodies. FIG. 7B shows antigen specific activation of T-cells
expressing either the scFv CD4 TAC (top) or scFV CAR (bottom).
T-cells expressing either the scFv CD4 TAC (top) or scFv CAR
(bottom) were incubated with plate bound Her2 antigen. Both
modified cells showed antigen specific activation. The DMSO
negative control showed no activity (Data not shown). FIG. 7C shows
comparable killing of MCF-7 human tumour cell line (Her2 positive)
by both scFv CD4 TAC and scFv CAR. Both scFv CD4 TAC and scFv CAR
were incubated with MCF-7 human tumour cell line (Her2 positive)
and compared to an empty vector control.
[0170] FIG. 8 is a characterization of CD4-TAC configuration 2.
FIG. 8A is a histogram of DARPin CD4-TAC configuration 2 relative
to vector control. Surface expression was probed with the FcHer2
modified antigen. Cells expressing CD4-TAC configuration 2 show a
distinct increase in FcHer2 binding demonstrating high surface
expression of the receptor. For clarity the model of CD4 TAC
configuration 2 is shown. FIG. 8B shows T-cells engineered with
DARPin TAC configuration 2 exposed to plate bound Her2 antigen.
Cytokine production and degranulation were measured. Data show that
DARPin TAC configuration 2 is a functional receptor. Treatment
without antigen showed no T-cell activation (data not shown). FIG.
8C shows growth of CD4 TAC configuration 2 relative to empty vector
control. Cells were grown in 100u/ml IL2 10 ng/ml IL7. Starting
with 100,000 cells, growth was monitored by counting culture
samples at predetermined intervals. Configuration 2 has a marked
reduced growth rate relative to control.
[0171] FIG. 9 shows the functionality of DARPin CD4 TAC
configuration 1. FIG. 9A shows surface expression of DARPin CD4 TAC
(red) compared to DARPin CAR (green), and the NGFR only control
(blue). Cells were probed with receptor specific antigen FcHer2.
Histogram shows that DARPin CD4 TAC is expressed well on the
surface. However, its maximal surface expression is lower compared
to the CAR construct. FIG. 9B shows growth of CD4 TAC configuration
1. For two weeks culture growth was monitored by sampling and
manually counting cells. The empty vector shows similar growth as
DARPin CAR. However TAC has reduced growth by comparison. FIGS. 9C
and 9D show the percentage of cells positive for various activation
and degradation markers. Empty vector, DARPin CD4 and DARPin CAR
were incubated with either plate bound antigen Her2 or DMSO
control. The results of three separate experiments are summarized
using the statistical analysis software SPICE. The scatter graph
shows the percentage of cells positive for a set of activation
markers. CD4-TAC show a higher percentage of cells, positive for
degranulation markers. DARPin CAR cells are positive for a variety
of activation markers with no significantly enriched population of
degranulation markers. The pie chart represents the same data. It
demonstrates that CD4-TAC has a markedly higher population of cells
focused on degranulation. CD4-TAC has a majority of activated cells
degranulate with various levels of cytokine production. However,
CARs show a more randomly distributed pattern of activation with
degranulation constituting less than 50% of the total population.
The pattern may be indicative of a less controlled T-cell
activation by CARs.
[0172] FIG. 10 shows cytotoxicity and overall activity of TAC and
CAR. Cells engineered with either TAC, CAR or empty vector control
were incubated with various human tumour cell lines. MDA MB 231, SK
OV 3 and A549 all express the Her2 antigen. LOXIMVI is Her2
negative. It was observed that in all cases, TAC shows enhanced
cytotoxicity. The antigen negative cell line LOCIMVI is not being
targeted, supporting that cytotoxicity is antigen specific.
[0173] FIG. 11 shows receptor surface expression and activation of
various TAC controls. Cell surface expression (left), degranulation
(middle) and cytokine production (right) is shown in FIG. 11A.
Constructs lacking specific domains were made to determine the
significance of these domains. From top to bottom the following
domains were removed: DARPin antigen binding domain and UCHT1 TCR
binding domain, with the full length TAC being at the bottom.
Surface expression of TAC without the UCHT1 domain resulted in
enhanced surface expression relative to full length CD4 TAC. The
DARPin negative mutant could not be detected using FcHer2 antigen.
Degranulation (middle) was only observed in full length TAC. Both
UCHT1 and DARPin deletion resulted in no degranulation. Similarly,
cytokine production was only observed in the full length TAC. FIG.
11B shows the mouse cell line D2F2 was engineered to express the
human Her2 antigen (D2F2/E2). Both cell lines were incubated with
T-cells engineered with full length CD4-TAC or its deletion
variants. The data show the Effector to Target ratio 4:1 endpoint.
Only full length CD4-TAC was able to elicit a cytotoxic response.
This demonstrates that DARPin and UCHT1 domains are involved in
receptor function.
[0174] FIG. 12 shows properties of various transmembrane TAC
variants. FIG. 12A is an overview of the various transmembrane
constructs. The first set of variants is lacking the cytosolic
domain. The CD4 TAC-cytosol has the entire cytosolic domain
removed. The synthetic construct has the CD4 TM replaced by a
designed, highly hydrophobic membrane domain. The glycophorine
variant replaces the CD4 transmembrane domain with the glycophorine
transmembrane domain. The GPI anchor variant uses the CD48 GPI
signal sequenced to attach the TAC to the membrane using the GPI
anchor. The CD8A TAC variant replaced the transmembrane and
cytosolic CD4 domain with the CD8a counterpart. FIG. 12B shows CD8
purified T-cells were engineered with the various constructs.
Surface expression of the various receptors relative to full length
TAC is shown. All data is relative to the median fluorescent
intensity of the control. All variants have a significantly lower
receptor surface expression compared to the full length CD4-TAC.
The GPI anchor TAC variant is not detectable above background. FIG.
12C depicts testing of the different variants for degranulation and
cytokine production. Cells were incubated with plate pound Her2
antigen. The activity is presented as percent of cells positive for
either the degranulation maker CD107a (left bar graph) or the
percent of all cytokine producing cells taken together (TNF.alpha.,
IFNg and TNF.alpha./IFNg, right bar graph). GPI anchored or CD8a
variants show background levels of degranulation and cytokine
production. Glycophorine, synthetic and -cytosol TAC variants show
a moderate level of degranulation and a low level of cytokine
production. In all cases the activity is well below full length
CD4-TAC. Taken together this shows that anchoring TAC without its
cytosolic domain leads to functional receptors with diminished
activity.
[0175] FIG. 13 shows Lck interaction with TAC variants. In FIG.
13A, Her2 antigen was covalently attached to magnetic beads. 293TM
cells were engineered to express both the TAC and TAC cytosolic
deletion variant as well as Lck. Beads were incubated with cell
lysates over night and subsequently washed and western blotted. Lck
was detected using an Lck antibody, TACs were detected via Myc
antibody. B-Actin was used as control. b-Actin was not pulled down
and only detected in the supernatant (S). However both full length
TAC and cytosolic deletion were efficiently pulled down and
detected in the pellet fraction (B). Vector control and TAC without
cytosolic domain show comparable levels of background Lck signal.
Full length CD4 TAC however shows a significant level of Lck
relative to the total amount. FIG. 13B shows densitometry analysis
of the Lck detected in the pellet. Signal was corrected relative to
the negative control. This data supports that Lck is able to
interact with full length CD4-TAC.
[0176] In FIG. 14, CD4 TAC surface expression and activity is
compared to a BiTE like variant. FIG. 14A shows NGFR only control
(left), CD4 TAC (middle) and BiTE like variant (right). Surface
expression was tested using the transduction marker NGFR and the
Her2 antigen. TAC shows much lower surface expression compared to
BiTEs. Most notably, BiTE seems to secrete enough coupling antibody
to enable transduction negative cells (NGFR-) to show strong
receptor expression. Both cytokine production and degranulation are
higher in BiTE like cells compared to TAG engineered cells. FIG.
14B compares cytotoxicity in various Her2 positive cell lines (MDA
MB 231, SK OV 3, A549). In contrast to cytokine production, TAG
engineered cells show significantly enhanced cytotoxic
activity.
[0177] FIG. 15 shows comparison of CD4 TAG WT to a random mutagen
library of UCHT1. To test the ability to change TAG properties, 24
amino acids found on the binding surface of UCHT1 and TCR epsilon
were individually mutated. This gives rise to a theoretical number
of 480 unique clones, all of which should be represented in this
random library. FIG. 15A shows the schematic representation of the
mutant. Markings indicate the mutations which are all in the
scFv-epsilon interface. Figure FSB is a histogram of surface
expression. Engineered cells were probed with FcHer2 antigen to
detect surface expressed receptor. The library shows a much
enhanced surface expression of the receptor. FIG. 15C shows WT and
Library CD4 TAG cells incubated with plate bound antigen. Their
ability to activate and produce cytokines is presented. The library
has similar activity compared to the WT. Without being bound by
theory, this supports the idea that expression properties of TAG
can be improved while retaining the original functional profile by
altering the scFv domain.
[0178] FIG. 16 shows enhanced surface expression of the A85V, T161P
mutant. The library was propagated for an extended period of time
to select for mutants with a growth advantage over the WT. A
selected mutant was analysed (A85V, T161P; numbering is based on
the UCHT1 domain fragment). FIG. 16A shows peripheral blood
mononuclear cells (PBMC) were engineered with either WT CD4-TAC or
the A85V, T161P mutant. The final CD4/CD8 populations between CD4
TAG (left) and A85V, T161P mutant (right) are compared. Notably, WT
CD4-TAC leads to a reduced population of CD4 positive cells. This
effect is not observed in the mutant cells. FIG. 16B shows surface
expression, as determined by NGFR transduction maker and FcHer2
positivity, and indicates enhanced surface expression of the A85V,
T161P mutant. FIG. 16C shows that A85V, T161P mutant cytokine
production is diminished (DMSO controls showed no activity, data
not shown). Degranulation between WT TAC and A85V, T161P mutant is
comparable.
[0179] FIG. 17 shows A85V, T161P mutant cytotoxicity and growth. In
FIG. 17A, T-cells engineered with WT CD4 TAC and A85V, T161P mutant
were incubated with the Her2 antigen positive cell lines SK OV 3,
MDA MB 231 and A549. In all cases, the mutant displayed a reduced
level of cytotoxicity; in the case of A549, no cytotoxicity was
detected. In FIG. 17B, cell growth in culture starting with 100 000
cells was monitored over 2 weeks. Periodically samples were taken
and cells were counted manually. The A85V, T161P mutant exhibits
markedly improved growth compared to the WT variant. Taken
together, this demonstrates that the library is likely to contain
various mutants that enable the modification and optimization of
several TAC functions. Thus, UCHT1 can be used as a functional
modulator.
[0180] While the present application has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the application is not
limited to the disclosed examples. To the contrary, the application
is intended to cover various modifications and equivalent
arrangements included within the spirit and scope of the appended
claims.
[0181] 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.
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Crystal structure of a complete ternary complex of T-cell
Sequence CWU 1
1
2511575DNAArtificial SequenceSynthetic 1atggatttcc aggtccagat
tttctccttc ctgctgattt ccgcaagcgt cattatgtca 60cggggctccg acctgggcaa
aaagctgctg gaggccgcta gggccgggca ggacgatgaa 120gtgagaatcc
tgatggccaa cggggctgac gtgaatgcta aggatgagta cggcctgacc
180cccctgtatc tggctacagc acacggccat ctggagatcg tggaagtcct
gctgaaaaac 240ggagccgacg tgaatgcagt cgatgccatt gggttcactc
ctctgcacct ggcagccttt 300atcggacatc tggagattgc agaagtgctg
ctgaagcacg gcgctgacgt gaacgcacag 360gataagttcg gaaaaaccgc
ttttgacatc agcattggca acggaaatga agacctggct 420gaaatcctgc
agaaactgaa tgaacagaaa ctgattagcg aagaagacct gaaccccggg
480ggaggaggag ggagcggggg aggaggcagc ggcgggggag gctctggagg
aggagggagc 540ggatccatgg acatccagat gactcagacc acaagctccc
tgtctgcaag tctgggcgac 600cgggtgacaa tctcctgcag agcctctcag
gatattagga actacctgaa ttggtatcag 660cagaaacctg atggcacagt
caagctgctg atctactata ccagccggct gcactcaggc 720gtgccaagca
aattctcagg aagcggctcc gggactgact actccctgac catctctaac
780ctggagcagg aagatattgc tacctatttc tgccagcagg gcaatacact
gccctggact 840tttgccggag gcaccaaact ggagatcaag gggggaggcg
ggagtggagg cgggggatca 900ggaggaggag gcagcggagg aggagggtcc
gaggtccagc tgcagcagag cggaccagaa 960ctggtgaagc ccggagcaag
tatgaaaatc tcctgtaagg cctcaggata cagcttcacc 1020ggctatacaa
tgaactgggt gaaacagtcc catggcaaga acctggaatg gatggggctg
1080attaatcctt acaaaggcgt cagcacctat aatcagaagt ttaaagacaa
ggccacactg 1140actgtggata agtctagttc aaccgcttac atggagctgc
tgtccctgac atctgaagac 1200agtgccgtgt actattgtgc tcggtctggc
tactatgggg acagtgattg gtacttcgat 1260gtctggggac agggcactac
cctgaccgtg ttttctacta gtggcggagg aggatcactc 1320gagagcggac
aggtgctgct ggaatccaat atcaaagtcc tgcccacttg gtctaccccc
1380gtgcagccta tggctctgat tgtgctggga ggagtcgcag gactgctgct
gtttatcggg 1440ctgggaattt tcttttgcgt gcgctgccgg caccggagaa
ggcaggccga gcgcatgagc 1500cagatcaagc gactgctgag cgagaagaaa
acctgtcagt gtccccatag attccagaag 1560acctgttcac ccatt
15752525PRTArtificial SequenceSynthetic 2Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala 20 25 30Ala Arg Ala Gly
Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly 35 40 45Ala Asp Val
Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu 50 55 60Ala Thr
Ala His Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn65 70 75
80Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His
85 90 95Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val Leu Leu
Lys 100 105 110His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
Thr Ala Phe 115 120 125Asp Ile Ser Ile Gly Asn Gly Asn Glu Asp Leu
Ala Glu Ile Leu Gln 130 135 140Lys Leu Asn Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu Asn Pro Gly145 150 155 160Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175Gly Gly Gly Ser
Gly Ser Met Asp Ile Gln Met Thr Gln Thr Thr Ser 180 185 190Ser Leu
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 195 200
205Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
210 215 220Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
Ser Gly225 230 235 240Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly
Thr Asp Tyr Ser Leu 245 250 255Thr Ile Ser Asn Leu Glu Gln Glu Asp
Ile Ala Thr Tyr Phe Cys Gln 260 265 270Gln Gly Asn Thr Leu Pro Trp
Thr Phe Ala Gly Gly Thr Lys Leu Glu 275 280 285Ile Lys Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 290 295 300Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu305 310 315
320Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly
325 330 335Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser
His Gly 340 345 350Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro Tyr
Lys Gly Val Ser 355 360 365Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala
Thr Leu Thr Val Asp Lys 370 375 380Ser Ser Ser Thr Ala Tyr Met Glu
Leu Leu Ser Leu Thr Ser Glu Asp385 390 395 400Ser Ala Val Tyr Tyr
Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp 405 410 415Trp Tyr Phe
Asp Val Trp Gly Gln Gly Thr Thr Leu Thr Val Phe Ser 420 425 430Thr
Ser Gly Gly Gly Gly Ser Leu Glu Ser Gly Gln Val Leu Leu Glu 435 440
445Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met
450 455 460Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe
Ile Gly465 470 475 480Leu Gly Ile Phe Phe Cys Val Arg Cys Arg His
Arg Arg Arg Gln Ala 485 490 495Glu Arg Met Ser Gln Ile Lys Arg Leu
Leu Ser Glu Lys Lys Thr Cys 500 505 510Gln Cys Pro His Arg Phe Gln
Lys Thr Cys Ser Pro Ile 515 520 52531647DNAArtificial
SequenceSynthetic 3atggactttc aggtgcagat tttctctttt ctgctgattt
ccgcaagcgt catcgagctc 60gggggggggg ggtcaggatc catggacatc cagatgactc
agaccacaag ctccctgagc 120gcatccctgg gcgaccgagt gacaatctca
tgcagagcca gccaggatat taggaactac 180ctgaattggt atcagcagaa
acctgacggc acagtcaagc tgctgatcta ctatacttcc 240cggctgcact
ctggcgtgcc aagtaaattc tctgggagtg gatcaggcac tgactactca
300ctgaccatca gcaacctgga gcaggaagat attgctacct atttctgcca
gcagggcaat 360acactgccct ggacttttgc aggcgggacc aaactggaga
tcaagggcgg cggcggaagt 420ggaggaggag gctcaggcgg aggagggagc
ggcggaggag gcagcgaggt ccagctgcag 480cagagcggac cagaactggt
gaagcctggc gcatccatga aaatctcttg taaggcctct 540gggtacagtt
tcaccggata tacaatgaac tgggtgaaac agtctcatgg caagaacctg
600gaatggatgg gcctgattaa tccttacaaa ggcgtcagca cctataatca
gaagtttaaa 660gacaaggcca cactgactgt ggataagtct agttcaaccg
cttacatgga gctgctgtca 720ctgacaagcg aagactccgc cgtgtactat
tgcgctagga gcggatacta tggcgactcc 780gattggtact tcgatgtctg
ggggcaggga actaccctga ccgtgtttag cactagtgga 840ggaggaggct
ctggaggagg agggagtgga ggcgggggat caggaggagg aggcagcgat
900atcatgtcac ggggctccga cctgggcaaa aagctgctgg aggccgctag
ggccgggcag 960gacgatgaag tgagaatcct gatggccaac ggggctgacg
tgaatgctaa ggatgagtac 1020ggcctgaccc ccctgtatct ggctacagca
cacggccatc tggagatcgt ggaagtcctg 1080ctgaaaaacg gagccgacgt
gaatgcagtc gatgccattg ggttcactcc tctgcacctg 1140gcagccttta
tcggacatct ggagattgca gaagtgctgc tgaagcacgg cgctgacgtg
1200aacgcacagg ataagttcgg aaaaaccgct tttgacatca gcattggcaa
cggaaatgaa 1260gacctggctg aaatcctgca gaaactgaat gaacagaaac
tgattagcga agaagacctg 1320aacgtcgacg gaggaggagg gtctggagga
gggggaagtg gcgggggagg cagcggggga 1380ggcgggtctc tcgagagtgg
ccaggtgctg ctggaaagca atatcaaggt cctgccaact 1440tggtccaccc
cagtgcagcc tatggctctg attgtgctgg gaggagtcgc aggactgctg
1500ctgtttatcg gcctggggat tttcttttgc gtgcgctgcc ggcaccggag
aaggcaggct 1560gagcgcatgt ctcagattaa gcgactgctg agcgagaaga
agacctgtca gtgcccccat 1620agattccaga aaacctgttc acccatt
16474549PRTArtificial SequenceSynthetic 4Met Asp Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Glu Leu Gly
Gly Gly Gly Ser Gly Ser Met Asp Ile Gln Met 20 25 30Thr Gln Thr Thr
Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr 35 40 45Ile Ser Cys
Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr 50 55 60Gln Gln
Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser65 70 75
80Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly
85 90 95Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile
Ala 100 105 110Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr
Phe Ala Gly 115 120 125Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly
Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Gln145 150 155 160Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala Ser Met Lys Ile Ser 165 170 175Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 180 185 190Lys Gln
Ser His Gly Lys Asn Leu Glu Trp Met Gly Leu Ile Asn Pro 195 200
205Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr
210 215 220Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu
Leu Ser225 230 235 240Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
Ala Arg Ser Gly Tyr 245 250 255Tyr Gly Asp Ser Asp Trp Tyr Phe Asp
Val Trp Gly Gln Gly Thr Thr 260 265 270Leu Thr Val Phe Ser Thr Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280 285Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Met Ser Arg 290 295 300Gly Ser Asp
Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln305 310 315
320Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala
325 330 335Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala Thr Ala
His Gly 340 345 350His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly
Ala Asp Val Asn 355 360 365Ala Val Asp Ala Ile Gly Phe Thr Pro Leu
His Leu Ala Ala Phe Ile 370 375 380Gly His Leu Glu Ile Ala Glu Val
Leu Leu Lys His Gly Ala Asp Val385 390 395 400Asn Ala Gln Asp Lys
Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Gly 405 410 415Asn Gly Asn
Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu Asn Glu Gln 420 425 430Lys
Leu Ile Ser Glu Glu Asp Leu Asn Val Asp Gly Gly Gly Gly Ser 435 440
445Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu
450 455 460Glu Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu
Pro Thr465 470 475 480Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile
Val Leu Gly Gly Val 485 490 495Ala Gly Leu Leu Leu Phe Ile Gly Leu
Gly Ile Phe Phe Cys Val Arg 500 505 510Cys Arg His Arg Arg Arg Gln
Ala Glu Arg Met Ser Gln Ile Lys Arg 515 520 525Leu Leu Ser Glu Lys
Lys Thr Cys Gln Cys Pro His Arg Phe Gln Lys 530 535 540Thr Cys Ser
Pro Ile545554DNAArtificial SequenceSynthetic 5atggatttcc aggtccagat
tttctccttc ctgctgattt ccgcaagcgt catt 54618PRTArtificial
SequenceSynthetic 6Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu
Ile Ser Ala Ser1 5 10 15Val Ile7387DNAArtificial SequenceSynthetic
7atgtcacggg gctccgacct gggcaaaaag ctgctggagg ccgctagggc cgggcaggac
60gatgaagtga gaatcctgat ggccaacggg gctgacgtga atgctaagga tgagtacggc
120ctgacccccc tgtatctggc tacagcacac ggccatctgg agatcgtgga
agtcctgctg 180aaaaacggag ccgacgtgaa tgcagtcgat gccattgggt
tcactcctct gcacctggca 240gcctttatcg gacatctgga gattgcagaa
gtgctgctga agcacggcgc tgacgtgaac 300gcacaggata agttcggaaa
aaccgctttt gacatcagca ttggcaacgg aaatgaagac 360ctggctgaaa
tcctgcagaa actgaat 3878129PRTArtificial SequenceSynthetic 8Met Ser
Arg Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg1 5 10 15Ala
Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 20 25
30Val Asn Ala Lys Asp Glu Tyr Gly Leu Thr Pro Leu Tyr Leu Ala Thr
35 40 45Ala His Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly
Ala 50 55 60Asp Val Asn Ala Val Asp Ala Ile Gly Phe Thr Pro Leu His
Leu Ala65 70 75 80Ala Phe Ile Gly His Leu Glu Ile Ala Glu Val Leu
Leu Lys His Gly 85 90 95Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys
Thr Ala Phe Asp Ile 100 105 110Ser Ile Gly Asn Gly Asn Glu Asp Leu
Ala Glu Ile Leu Gln Lys Leu 115 120 125Asn930DNAArtificial
SequenceSynthetic 9gaacagaaac tgattagcga agaagacctg
301010PRTArtificial SequenceSynthetic 10Glu Gln Lys Leu Ile Ser Glu
Glu Asp Leu1 5 101175DNAArtificial SequenceSynthetic 11aaccccgggg
gaggaggagg gagcggggga ggaggcagcg gcgggggagg ctctggagga 60ggagggagcg
gatcc 751225PRTArtificial SequenceSynthetic 12Asn Pro Gly Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly1 5 10 15Gly Ser Gly Gly
Gly Gly Ser Gly Ser 20 2513750DNAArtificial SequenceSynthetic
13atggacatcc agatgactca gaccacaagc tccctgtctg caagtctggg cgaccgggtg
60acaatctcct gcagagcctc tcaggatatt aggaactacc tgaattggta tcagcagaaa
120cctgatggca cagtcaagct gctgatctac tataccagcc ggctgcactc
aggcgtgcca 180agcaaattct caggaagcgg ctccgggact gactactccc
tgaccatctc taacctggag 240caggaagata ttgctaccta tttctgccag
cagggcaata cactgccctg gacttttgcc 300ggaggcacca aactggagat
caagggggga ggcgggagtg gaggcggggg atcaggagga 360ggaggcagcg
gaggaggagg gtccgaggtc cagctgcagc agagcggacc agaactggtg
420aagcccggag caagtatgaa aatctcctgt aaggcctcag gatacagctt
caccggctat 480acaatgaact gggtgaaaca gtcccatggc aagaacctgg
aatggatggg gctgattaat 540ccttacaaag gcgtcagcac ctataatcag
aagtttaaag acaaggccac actgactgtg 600gataagtcta gttcaaccgc
ttacatggag ctgctgtccc tgacatctga agacagtgcc 660gtgtactatt
gtgctcggtc tggctactat ggggacagtg attggtactt cgatgtctgg
720ggacagggca ctaccctgac cgtgttttct 75014250PRTArtificial
SequenceSynthetic 14Met Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
Ser Ala Ser Leu1 5 10 15Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln Asp Ile Arg Asn 20 25 30Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr Val Lys Leu Leu 35 40 45Ile Tyr Tyr Thr Ser Arg Leu His Ser
Gly Val Pro Ser Lys Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Tyr
Ser Leu Thr Ile Ser Asn Leu Glu65 70 75 80Gln Glu Asp Ile Ala Thr
Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95Trp Thr Phe Ala Gly
Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly 100 105 110Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 130 135
140Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly
Tyr145 150 155 160Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn
Leu Glu Trp Met 165 170 175Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser
Thr Tyr Asn Gln Lys Phe 180 185 190Lys Asp Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Ser Thr Ala Tyr 195 200 205Met Glu Leu Leu Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 210 215 220Ala Arg Ser Gly
Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp225 230 235 240Gly
Gln Gly Thr Thr Leu Thr Val Phe Ser 245 2501527DNAArtificial
SequenceSynthetic 15actagtggcg gaggaggatc actcgag
27169PRTArtificial SequenceSynthetic 16Thr Ser Gly Gly Gly Gly Ser
Leu Glu1 517252DNAArtificial SequenceSynthetic 17agcggacagg
tgctgctgga atccaatatc aaagtcctgc ccacttggtc tacccccgtg 60cagcctatgg
ctctgattgt gctgggagga gtcgcaggac tgctgctgtt tatcgggctg
120ggaattttct tttgcgtgcg ctgccggcac cggagaaggc aggccgagcg
catgagccag 180atcaagcgac tgctgagcga gaagaaaacc tgtcagtgtc
cccatagatt ccagaagacc 240tgttcaccca tt 2521884PRTArtificial
SequenceSynthetic 18Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val
Leu Pro Thr Trp1 5 10 15Ser Thr Pro Val Gln Pro Met Ala Leu Ile Val
Leu Gly Gly Val Ala 20 25
30Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys
35 40 45Arg His Arg Arg Arg Gln Ala Glu Arg Met Ser Gln Ile Lys Arg
Leu 50 55 60Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro His Arg Phe Gln
Lys Thr65 70 75 80Cys Ser Pro Ile1956PRTArtificial
SequenceSynthetic 19Gly Gly Gly Gly Ser Ala Glu Ala Ala Ala Lys Glu
Ala Ala Ala Lys1 5 10 15Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala
Leu Glu Ala Glu Ala 20 25 30Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
Ala Ala Lys Glu Ala Ala 35 40 45Ala Lys Ala Gly Gly Gly Gly Ser 50
552055PRTArtificial SequenceSynthetic 20Gly Gly Gly Gly Ser Ala Ala
Leu Ser Pro Ser Pro Leu Ala Pro Gly1 5 10 15Pro Ala Ala Pro Ala Ala
Leu Ala Pro Ala Pro Leu Ala Pro Gly Pro 20 25 30Ser Ala Pro Ala Ala
Ala Ser Pro Ser Pro Leu Ala Pro Gly Pro Ser 35 40 45Ala Pro Gly Gly
Gly Gly Ser 50 552166PRTArtificial SequenceSynthetic 21Ser Gly Ala
Val Leu Leu Ala Ser Ala Val Ala Val Leu Pro Ser Ala1 5 10 15Ser Ser
Pro Val Ala Pro Ser Gly Ala Val Leu Leu Ala Ser Ala Val 20 25 30Ala
Val Leu Pro Ser Ala Ser Ser Pro Val Ala Pro Ser Gly Ala Val 35 40
45Leu Leu Ala Ser Ala Val Ala Val Leu Pro Ser Ala Ser Ser Pro Val
50 55 60Ala Pro6522729DNAArtificial SequenceSynthetic 22atgtctagac
aggtacaact gcagcagtca ggacctgaac tgaagaagcc tggagagaca 60gtcaagatct
cctgcaaggc ctctgggtat cctttcacaa actatggaat gaactgggtg
120aagcaggctc caggacaggg tttaaagtgg atgggctgga ttaacacctc
cactggagag 180tcaacatttg ctgatgactt caagggacgg tttgacttct
ctttggaaac ctctgccaac 240actgcctatt tgcagatcaa caacctcaaa
agtgaagaca tggctacata tttctgtgca 300agatgggagg tttaccacgg
ctacgttcct tactggggcc aagggaccac ggtcaccgtt 360tcctctggcg
gtggcggttc tggtggcggt ggctccggcg gtggcggttc tgacatccag
420ctgacccagt ctcacaaatt cctgtccact tcagtaggag acagggtcag
catcacctgc 480aaggccagtc aggatgtgta taatgctgtt gcctggtatc
aacagaaacc aggacaatct 540cctaaacttc tgatttactc ggcatcctcc
cggtacactg gagtcccttc tcgcttcact 600ggcagtggct ctgggccgga
tttcactttc accatcagca gtgtgcaggc tgaagacctg 660gcagtttatt
tctgtcagca acattttcgt actccattca cgttcggctc ggggacaaaa 720ttggagatc
72923243PRTArtificial SequenceSynthetic 23Met Ser Arg Gln Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys1 5 10 15Pro Gly Glu Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Pro Phe 20 25 30Thr Asn Tyr Gly
Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 35 40 45Lys Trp Met
Gly Trp Ile Asn Thr Ser Thr Gly Glu Ser Thr Phe Ala 50 55 60Asp Asp
Phe Lys Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala Asn65 70 75
80Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp Met Ala Thr
85 90 95Tyr Phe Cys Ala Arg Trp Glu Val Tyr His Gly Tyr Val Pro Tyr
Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
Gln Leu Thr Gln Ser 130 135 140His Lys Phe Leu Ser Thr Ser Val Gly
Asp Arg Val Ser Ile Thr Cys145 150 155 160Lys Ala Ser Gln Asp Val
Tyr Asn Ala Val Ala Trp Tyr Gln Gln Lys 165 170 175Pro Gly Gln Ser
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Arg Tyr 180 185 190Thr Gly
Val Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Pro Asp Phe 195 200
205Thr Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Phe
210 215 220Cys Gln Gln His Phe Arg Thr Pro Phe Thr Phe Gly Ser Gly
Thr Lys225 230 235 240Leu Glu Ile24750DNAArtificial
SequenceSynthetic 24atggacatcc agatgactca gaccacaagc tccctgtctg
caagtctggg cgaccgggtg 60acaatctcct gcagagcctc tcaggatatt aggaactacc
tgaattggta tcagcagaaa 120cctgatggca cagtcaagct gctgatctac
tataccagcc ggctgcactc aggcgtgcca 180agcaaattct caggaagcgg
ctccgggact gactactccc tgaccatctc taacctggag 240caggaagata
ttgttaccta tttctgccag cagggcaata cactgccctg gacttttgcc
300ggaggcacca aactggagat caagggggga ggcgggagtg gaggcggggg
atcaggagga 360ggaggcagcg gaggaggagg gtccgaggtc cagctgcagc
agagcggacc agaactggtg 420aagcccggag caagtatgaa aatctcctgt
aaggcctcag gatacagctt caccggctat 480ccgatgaact gggtgaaaca
gtcccatggc aagaacctgg aatggatggg gctgattaat 540ccttacaaag
gcgtcagcac ctataatcag aagtttaaag acaaggccac actgactgtg
600gataagtcta gttcaaccgc ttacatggag ctgctgtccc tgacatctga
agacagtgcc 660gtgtactatt gtgctcggtc tggctactat ggggacagtg
attggtactt cgatgtctgg 720ggacagggca ctaccctgac cgtgttttct
75025250PRTArtificial SequenceSynthetic 25Met Asp Ile Gln Met Thr
Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu1 5 10 15Gly Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn 20 25 30Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu 35 40 45Ile Tyr Tyr
Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu65 70 75
80Gln Glu Asp Ile Val Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
85 90 95Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly
Gly 100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 115 120 125Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 130 135 140Ser Met Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Gly Tyr145 150 155 160Pro Met Asn Trp Val Lys
Gln Ser His Gly Lys Asn Leu Glu Trp Met 165 170 175Gly Leu Ile Asn
Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 180 185 190Lys Asp
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 195 200
205Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
210 215 220Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp
Val Trp225 230 235 240Gly Gln Gly Thr Thr Leu Thr Val Phe Ser 245
250
* * * * *