U.S. patent application number 17/664731 was filed with the patent office on 2022-09-22 for engineered t cells and uses therefor.
The applicant listed for this patent is Pieris Pharmaceuticals GmbH. Invention is credited to Marlon Hinner.
Application Number | 20220296690 17/664731 |
Document ID | / |
Family ID | 1000006388083 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220296690 |
Kind Code |
A1 |
Hinner; Marlon |
September 22, 2022 |
ENGINEERED T CELLS AND USES THEREFOR
Abstract
Lipocalin muteins specific to a predetermined antigen can be
transduced into a T cell to bring therapeutic benefits to patients
in need. In one example, a lipocalin mutein specific to a
predetermined antigen (e.g., a target differentially expressed on
the surface of a tumor cell) can be transduced into a T cell
membrane to serve as an antigen receptor, offering benefits over
conventionally deployed antibody-derived protein moieties such as a
single chain variable fragment (scFv). Benefits include a more
stable structure, leading to superior target engagement, for
example. Further, lipocalin muteins specific to a predetermined
antigen (e.g. an immunomodulatory target such as an immune
checkpoint or costimulatory molecule) can be transduced into a T
cell for secretion thereby, bringing an added therapeutic benefit.
Specific examples of such modified T cells and methods of making
and using the same are provided herein.
Inventors: |
Hinner; Marlon; (Munich,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pieris Pharmaceuticals GmbH |
Freising-Weihenstephan |
|
DE |
|
|
Family ID: |
1000006388083 |
Appl. No.: |
17/664731 |
Filed: |
May 24, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15542915 |
Jul 11, 2017 |
11382963 |
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PCT/EP2016/050326 |
Jan 11, 2016 |
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17664731 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/02 20130101;
A61K 2039/5156 20130101; C07K 2319/22 20130101; C12N 5/0636
20130101; C07K 2319/33 20130101; A61K 39/0011 20130101; C07K
14/70521 20130101; C07K 14/5434 20130101; C12N 2510/00 20130101;
C07K 14/70596 20130101; C07K 14/47 20130101; C07K 14/705 20130101;
C07K 2318/20 20130101; C07K 2317/622 20130101; C07K 2319/03
20130101; C07K 14/7051 20130101; A61K 2039/5158 20130101; C07K
2319/74 20130101; C07K 2319/30 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/705 20060101 C07K014/705; C12N 5/0783 20060101
C12N005/0783; C07K 14/725 20060101 C07K014/725; C07K 14/47 20060101
C07K014/47; C07K 14/54 20060101 C07K014/54 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 2015 |
EP |
15000047.9 |
Claims
1. A modified T cell expressing and secreting a specific binding
polypeptide comprising a lipocalin mutein selected from the group
consisting of a mutein of human tear lipocalin (hTlc) and a mutein
of human neutrophil gelatinase-associated lipocalin (hNGAL),
wherein the lipocalin mutein is modified such that it is specific
for a target other than the natural target of hTlc and hNGAL,
respectively.
2. The modified T cell of claim 1, wherein the modified T cell
secretes the hTlc or the hNGAL mutein upon activation.
3. The modified T cell of claim 1, wherein the lipocalin mutein is
an hNGAL mutein.
4. The modified T cell of claim 3, wherein the lipocalin mutein is
an hNGAL mutein having at least 80% sequence identity to mature,
wild-type hNGAL (SEQ ID NO: 30).
5. The modified T cell of claim 4, wherein the hNGAL mutein retains
a supersecondary or tertiary structure of mature, wild-type hNGAL
(SEQ ID NO: 30).
6. The modified T cell of claim 4, wherein the hNGAL mutein
comprises an amino acid substitution at each of A40, Q49, L70, R72,
K73, D77, W79, R81, L103, K125, S127, and Y132 relative to mature,
wild-type hNGAL (SEQ ID NO: 30).
7. The modified T cell of claim 4, wherein the hNGAL mutein
comprises the amino acid sequence of mature, wild-type hNGAL (SEQ
ID NO: 30) but for from 13 to 26 amino acid substitutions relative
to mature, wild-type hNGAL, wherein at least A40, Q49, L70, R72,
K73, D77, W79, R81, C87, L103, K125, S127, and Y132 are replaced
with a different amino acid residue.
8. The modified T cell of claim 7, wherein the hNGAL mutein further
comprises at least one additional amino acid replacement at a
position selected from E44, K46, D47, K50, N65, F71, P101, G102,
T104, V126, Q128, R130, and K134 relative to mature, wild-type
hNGAL (SEQ ID NO: 30).
9. The modified T cell of claim 1, wherein the lipocalin mutein is
an hNGAL mutein having at least 80% sequence identity to SEQ ID NO:
3.
10. The modified T cell of claim 9, wherein the hNGAL mutein
comprises an amino acid substitution at each of A40, Q49, L70, R72,
K73, D77, W79, R81, L103, K125, S127, and Y132 relative to mature,
wild-type hNGAL (SEQ ID NO: 30).
11. The modified T cell of claim 9, wherein the hNGAL mutein
comprises the amino acid sequence of mature, wild-type hNGAL (SEQ
ID NO: 30) but for from 13 to 26 amino acid substitutions relative
to mature, wild-type hNGAL, wherein at least A40, Q49, L70, R72,
K73, D77, W79, R81, C87, L103, K125, S127, and Y132 are replaced
with a different amino acid residue.
12. The modified T cell of claim 1, wherein the modified T cell
further comprises a chimeric antigen receptor.
13. The modified T cell of claim 12, wherein the chimeric antigen
receptor comprises: i. an extracellular ligand binding domain; ii.
at least one costimulatory domain selected from a CD28 domain, a
CD137 domain, an ICOS domain, an OX40 domain, a CD27 domain, and a
CD40 domain; and iii. a cytoplasmic domain comprising at least one
intracellular signaling domain, wherein the at least one
intracellular signaling domain comprises a CD3zeta signaling
domain.
14. A modified T cell comprising: (a) single-domain, monomeric
polypeptide comprising a mutein of human neutrophil
gelatinase-associated lipocalin (hNGAL), wherein the hNGAL mutein
has at least 80% sequence identity to mature, wild-type hNGAL (SEQ
ID NO: 30); and (b) a chimeric antigen receptor.
15. The modified T cell of claim 14, wherein the chimeric antigen
receptor comprises: i. an extracellular ligand binding domain; ii.
at least one costimulatory domain selected from a CD28 domain, a
CD137 domain, an ICOS domain, an OX40 domain, a CD27 domain, and a
CD40 domain; and iii. a cytoplasmic domain comprising at least one
intracellular signaling domain, wherein the at least one
intracellular signaling domain comprises a CD3zeta signaling
domain.
16. A pharmaceutical composition comprising a modified T cell of
claim 1 and optionally a pharmaceutically acceptable carrier or
excipient.
17. A pharmaceutical composition comprising a modified T cell of
claim 14 and optionally a pharmaceutically acceptable carrier or
excipient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/542,915, filed Jul. 11, 2017 as a National Stage application
of PCT/EP2016/050326, filed Jan. 11, 2016, which claims priority
from European application EP 15000047.9, filed Jan. 12, 2015. The
entire contents of each of the aforementioned applications are
incorporated by reference in their entireties.
BACKGROUND
[0002] T-cells transduced with artificial chimeric antigen
receptors (CARs), also called CAR-T cells, are being deployed for
the treatment of cancer (Shi et al. 2014). The CAR-T approach has
provided positive clinical results in hematologic cancers such as
acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia
(CLL). In a recent trial, for example, 30 patients with relapsed or
refractory ALL were infused with autologous T cells transduced with
the CD19-directed chimeric antigen receptor CTL019 (formerly called
CART-19). Of these, 27 patients (90%) achieved complete remission
(Maude et al. 2014). Sustained remission was achieved with a
6-month event-free survival rate of 67% and an overall survival
rate of 78%.
[0003] CARs are transmembrane proteins stably anchored in the
T-cell plasma membrane. A CAR is an artificial fusion of multiple
parts: the extracellular part consists of a recognition domain
binding to a cancer cell target; the intracellular part contains
the signaling domain(s) of one or more (co-)stimulatory
immunoreceptors; and the two are fused via a linker- and
transmembrane domain. In the case of CTL019, for example, the
recognition domain is an antibody single chain fragment (scFv)
specific for CD19, the linker- and transmembrane regions are
grafted from the membrane protein CD8, and the intracellular
signaling part consists of the complete intracellular domains of
CD137 and CD3zeta fused in tandem. When a T-cell transduced with
this construct encounters a CD19-positive target cell, the chimeric
antigen receptor is clustered, which results in activation of the
signaling pathways downstream of CD3zeta and of the costimulatory
receptor CD137, in turn leading to activation of T-cell
proliferation, cytokine secretion, survival and the capacity to
kill.
[0004] With this design, CTL019 is an example for a "second
generation" CAR, identified by the presence of two
immunostimulatory domains. In contrast, "first generation" CARs
contain only a single immunostimulatory domain--usually that of
CD3zeta--while in "third generation" CARs, overall three
intracellular immunostimulatory domains are fused in tandem, for
example those of CD3zeta, CD28 and CD137. First generation CARs
have failed to show efficacy in the clinic--potentially due to the
lack of activity and long-term persistence of the CAR-T cells in
patients (Kershaw et al. 2006)--while third generation CARs have
yet to show their utility in a clinical setting.
[0005] To date, the extracellular recognition domains of CARs are
almost exclusively scFv's (with the few exceptions instead
utilizing the extracellular domains of ligands or receptors), which
is due to the fact that a single-chain functional protein (or
monomer) is required to facilitate a straightforward fusion design.
Accordingly, a tetrameric mAb or even a dimeric Fab would present
severe challenges as an extracellular recognition domain.
[0006] Although successes have been demonstrated with various scFv
approaches, that construct is an artificial fusion protein of the
variable regions of the heavy (VH) and light chains (VL) of an
antibody, connected with a short linker peptide of ten to about 25
amino acids. This artificial design leads to scFv-specific problems
that are well known in the art. Indeed, scFv's typically display
poor biophysical and developability behavior, such as low
stability, bad expression yields (Haisma et al. 1998a; Haisma et
al. 1998b, Peipp et al. 2004), the potential failure to be
efficiently secreted in mammalian cells (Brocks et al. 1997) and,
importantly, an oftentimes strong tendency to aggregate (see e.g.
Ewert et al. 2003a; Ewert et al. 2003b; Schaefer et al. 2012).
[0007] When an scFv is used for the construction of a CAR, these
properties can be deleterious at multiple stages. First, the
generation of a well-behaved scFv is difficult, and may require
extensive optimization and engineering. Second, in the
scFv-containing CAR, poor biophysical behavior can lead to poor and
potentially unpredictable cell surface expression, reducing the
number of CARs available to bind to a target antigen. Third, the
tendency of an scFv to aggregate may lead to an undesired
clustering of CARs on the T-cell surface, consequently leading to
an undesired spurious activation of the T-cell in the absence of
any specific target-bearing cell. The latter two issues can be a
serious threat to the efficacy and safety of any given CAR,
presenting clear challenges to obtain a predictable and
well-behaved CAR-T system.
[0008] The use of a polypeptide that employs a naturally occurring
secondary and tertiary structure could provide pronounced benefits
to an scFv approach. A mutein of a lipocalin that binds to a
desired tumor target is an example of a suitable alternative. A
lipocalin mutein, or Anticalin.RTM., is an engineered binding
protein based on a lipocalin (preferably of human origin) such as
Tear Lipocalin (TLc) or NGAL. Unlike an antibody, an Anticalin is a
single-domain protein; and unlike an scFv, an Anticalin is
single-domain by nature, therefore per se a well-behaved monomer
from a biophysical property standpoint. Apart from exhibiting
superior biophysical properties, Anticalins provide tunable
affinities from nanomolar to low picomolar range, while having a
demonstrated ability to engage targets across a broad spectrum,
including immune checkpoints.
[0009] Another application of a CAR-T involves engineering these
cells with the capacity to secrete proinflammatory cytokines. In
this vein, the engineered CAR-T can lead to accumulation of a
proinflammatory cytokine in the tumor micronenvironment where the
CAR-T traffics. A proinflammatory cytokine may be desired to
facilitate recruiting a second wave of immune cells in a locally
restricted fashion to initiate a more complete and potentially
target-independent attack of the cells of the tumor. This approach,
as recently reviewed (Chmielewski et al. 2014), has been described
in particular utilizing an engineered single-chain variant of
Interleukin 12, thereafter called scIL-12. Here, secretion of
scIL-12 can either be constitutive or be coupled to activation of
the T-cell via the CAR. The latter approach has in some instances
been termed TRUCK ("T-cells redirected for universal cytokine
killing"), which is why in the following the approach and related
approaches are termed "TRUCK-like". A TRUCK-like approach with
constitutive scIL-12 expression has already been brought to the
clinic in two separate trials, which currently are suspended
(NCT01236573) or terminated (NCT01457131), respectively.
Potentially, constitutive expression of scIL-12 was not
sufficiently localized to lead to the desired effects, or led to
intolerable toxicity.
[0010] To improve localized scIL-12 secretion, CAR-T cells have
been transduced with the scIL-12 construct expressed under control
of the nuclear-factor of the activated T-cell (NFAT)-derived
minimal promoter to initiate IL-12 transcription upon signaling of
the TCR/CD3 pathway (Chmielewski et al. 2011b; Zhang et al. 2011).
Other promoters sensitive to signals of the same pathway, like the
IL-2 promoter (Jaalouk et al. 2006) may also be feasible and could
allow tuning the produced amount and the selectivity of cytokine
production.
[0011] Using inducible scIL-12 expression, improved tumor reduction
was observed compared to unmodified CAR-T cells in preclinical
models (Chmielewski et al. 2011b; Zhang et al. 2011; Pegram et al.
2012; Pegram et al. 2014). Other cytokines, such as IL15, IL-18,
IL-23, IL-27 and IFN-gamma, and other producer cells, such as NK
cells, have been proposed for the approach. (Chmielewski et al.
2014).
[0012] The ability to engineer a T-Cell to secrete yet other types
of therapeutic proteins upon T-Cell activation would be desirable,
further expanding the repertoire to therapeutic modalities
available in the tumor microenvironment. Particularly desirable
would be specific binding proteins, e.g. a monoclonal antibody,
that could bind to an immunomodulatory target, such as an immune
checkpoint like CTLA4 or PD1, or to a costimulatory target such as
CD137 or OX40. There are at least two drawbacks to using an
antibody in this setting, however. First, antibodies are extremely
large, tetrameric proteins, limiting the ability of a T-Cell to
produce and secrete a functional antibody. Second, antibodies
typically have a half-life in man of two to three weeks, which
limits their application in an immunomodulatory approach, given
potential safety concerns.
[0013] A smaller, more stable monomeric polypeptide having a
shorter half-life could overcome these two shortcomings of
antibodies. An example for such a molecule would be a mutein of a
lipocalin that is specific for an immunomodulatory target and is
monomeric, stable and has a half-life tunable from hours to days in
man and a molecular weight as low as 18 kilodalton.
DETAILED DESCRIPTION
[0014] In one aspect, the disclosure provides a CAR that contains a
polypeptide defining a naturally occurring monomeric motif (e.g. a
lipocalin mutein) specific to a predetermined antigen (e.g., a
target differentially expressed on the surface of a tumor cell)
that is transduced into a T cell membrane to serve as an antigen
receptor, offering benefits over conventionally deployed
antibody-derived protein moieties such as a single chain variable
fragment (scFv). Benefits include a more stable structure, leading
to superior target engagement, for example. As used herein, a
"naturally occurring motif" is hereby defined as a motif that can
be found endogenously in a reference species, such as human; for
example, a lipocalin is a naturally occurring motif (as is a
lipocalin mutein that maintains the supersecondary or tertiary
structure of a lipocalin), whereas a polypeptide such as a scFv is
not a naturally occurring motif. Accordingly, "motif" when used
herein in the context of a "naturally occurring motif" is
preferably a naturally-occurring structure, preferably a
polypeptide. Said structure preferably exists endogenously in a
reference species. The skilled worker is able to readily determine
whether an engineered polypeptide defines such a naturally
occurring motif.
[0015] In a preferred embodiment a polypeptide defining a naturally
occurring monomeric motif is not a scFv. However, that being so, a
polypeptide defining a naturally occurring monomeric motif may
comprise a scFv, however, in addition to a further monomeric
polypeptide which is not a scFv. For example, a polypeptide
defining a naturally occurring monomeric motif may comprise a scFv
and a lipocalin, e.g. fused by a spacer, such as a linker amino
acid sequence. Both the scFv and the further monomeric polypeptide
which is not a scFV, may be specific to the same predetermined
antigen or may be specific for different predetermined targets.
This means that the scFv binds to a predetermined target that is
different from the predetermined target to which the further
monomeric polypeptide which, however, is not a scFv, binds to.
[0016] When referred to herein a "polypeptide that defines a
naturally occurring motif" includes that such a polypeptide may be
modified (engineered) to be able to bind to a target associated
with a tumor. For example, a lipocalin, that is within the context
of the present invention a polypeptide that defines a naturally
occurring motif, binds to its natural target such as lysozyme in
case of tear lipocalin (TLC) or iron in case of LCN2/NGAL. However,
such lipocalins can be modified such that they bind to a target
other than their natural target. Hence, the term "naturally
occurring motif" includes, that a polypeptide defining such a
naturally occurring motif that can be found endogenously in a
reference species, can preferably be modified or engineered to bind
to a target associated with a tumor. Yet, the modification is
preferably not a modification resulting in the generation of a
scFv.
[0017] The skilled worker will appreciate that the following
elements are to be considered when designing a CAR: [0018] (i) The
CAR primary sequence, consisting of a fusion of the binding
element, linker region, transmembrane domain and intracellular
signaling domains [0019] (ii) The method used to transduce a T-cell
[0020] (iii) Suitable assays such as an in vitro assay, to test the
functionality of the CAR [0021] (iv) In-vivo assays to test the
functionality of the CAR Several designs are possible for such a
CAR, with the following to be regarded as a non-limiting example.
The tumor-targeting moiety of a CAR-T may comprise a mutein of a
polypeptide that, endogenously, defines a monomeric polypeptide,
such as a lipocalin. The target to which such mutein (e.g., an
Anticalin) may bind includes CAIX, CD19, CD20, CD22, CD30, CD33,
CD44v7/8, CEA, EGP-2, EGP-40, FBP, Fetal acethylcholine receptor,
GD2, GD3, Her2/neu, IL-13R-a2, KDR, k-light chain, L1 cell adhesion
molecule, LeY, MAGE-A1, Mesothelin, MUC1, NKG2D ligands, Oncofetal
antigen (h5T4), PSCA, PSMA, TAG-72, VEGF-R2, the .alpha.-Folate
receptor and others. Other tumor-associated targets that might be
considered of interest in this context include EGFR, EPHA2,
melanoma 30 associated chondroitin sulfate proteoglycan, IGFR1,
fibroblast-activating protein alpha, c-MET, EpCAM, and GPC-3.
Further, targets not directly expressed on the tumor cell surface,
but associated with the tumor stroma, such as VEGF-A or Ang2, are
suitable.
[0022] The linker region and transmembrane domain have the function
of stably anchoring the CAR in the cell membrane, while providing
for a certain distance between the membrane and the binding element
to allow for an effective and activatory formation of the
CAR/target complex upon an encounter of the T-cell and the target
cell. Typical linker regions described in published examples lead
to dimerization of the CAR. When such linker domains are used, the
cytoplasmic tail of the dimeric CAR therefore contains two copies
of the respective intracellular signaling regions, copying what is
found in nature for the T-cell receptor (2 copies of CD3zeta) and
the homodimeric receptor CD28. SEQ ID NO: 4 represents the amino
acid sequence of one such example, based on a linker region and the
transmembrane domain of CD8 alpha (cf. WO2014011988 A2; for all SEQ
ID NO's of this application, one of the possible DNA sequences
encoding the respective amino acid sequence is provided below as
detailed in Table 1).
[0023] As described to date, a CAR may contain up to three
intracellular signaling domains in tandem, one of which being a
full intracellular signaling domain of CD3zeta (SEQ ID NO: 5) and
located at the C-terminus. In second-generation CARs, an additional
costimulatory signaling domain is inserted between the
transmembrane domain and the CD3zeta intracellular domain; for
example, the full intracellular domain of CD28 (SEQ ID NO: 6) or
that of CD137 (SEQ ID NO: 7). Alternatively, the intracellular
signaling domain of ICOS or of other costimulatory receptors from
the TNFR family, including but not limited to Ox-40, CD27 or CD40,
may also be employed. In third-generation CARs, three of these
costimulatory domains can be combined, with a typical example
consisting of the combination CD137, CD28 and CD3zeta. A mutein of
a polypeptide that, endogenously, defines a monomeric polypeptide
(such as a lipocalin) can be employed in a first, second, third or
future generation CAR.
[0024] The tumor-binding moiety may, for example, be an Anticalin
specific for a target selected from the group consisting of c-Met
and GPC-3. The sequence of such an Anticalin binding c-Met is
provided by SEQ ID NO: 1, while the sequence of an Anticalin
binding GPC-3 is provided by SEQ ID NO: 2. As the linker and
transmembrane region, the CD8 linker and transmembrane region (SEQ
ID NO: 4) may be utilized. For intracellular signalling, the
intracellular signaling domain of CD3zeta (SEQ ID NO: 5), either in
tandem with CD28 (SEQ ID NO: 6) or CD137 (SEQ ID NO: 7), can be
employed. The resulting four constructs, accordingly, could be:
c-Met-CD28-CD3zeta (SEQ ID NO: 8), c-Met-CD137-CD3zeta (SEQ ID NO:
9), GPC3-CD28-CD3zeta (SEQ ID NO: 10) and GPC3-CD137-CD3zeta (SEQ
ID NO: 11).
[0025] A number of methods have been devised to genetically modify
lymphocytes ex vivo to overexpress CARs. Retroviral vectors are
established and currently widely used, allowing for permanent and
heritable CAR expression due to their integration into genomic DNA.
For example, retroviral vectors derived from gamma-retrovirus have
been utilized for lymphocyte gene transfer in clinical applications
since 1990 (Rosenberg et al. 1990). As an alternative, an HIV-based
lentiviral vector may provide advantages such as higher and more
stable expression of the transgene, and potentially increased
safety compared to gamma-retroviral vectors. Other possible methods
for gene transfer include electroporation of mRNA constructs, if
CAR expression is desired to be transient only (Birkholz et al.
2009); (Zhao et al. 2006), and transposon-based systems such as
"piggybac" and "sleeping beauty" (Maiti et al. 2013). A
retrovirus-based system based on the vector pBULLET, as described
in Willemsen et al. 2000, is utilized in this application to
express SEQ ID NOs: 8-11. Alternatively, pSTITCH (Weijtens et al.
1998) also may be employed.
[0026] One or more in vitro assays can be employed to test the
functionality of a CAR, using standard methods that serve to
demonstrate the activation of T-cells. Investigations include the
responses of T-cells to activation, namely proliferation and
prolonged survival, the production of cytokines such as IL-2 and
IFN-gamma, and the capacity to kill target cells. The latter can be
determined, e.g., by direct observation of target cell killing, but
also by indirect methods that show for example the release of
intracellular components of the target cell or the generation of
cytotoxic molecules by the T-cells.
[0027] To investigate the functionality of a given CAR-design in
vivo, mouse models may be employed.
[0028] Examples for both in-vitro and in-vivo assays are provided
in the literature, cf. for example Hombach et al. 2010; Chmielewski
et al. 2011a; Chmielewski et al. 2011b; Kofler et al. 2011;
Chmielewski et al. 2012; Maliar et al. 2012; Pegram et al. 2012;
Chmielewski et al. 2013a; Chmielewski et al. 2013b; Hombach et al.
2013; Pegram et al. 2014; and Textor et al. 2014.
[0029] The skilled worker also will appreciate that beyond a
lipocalin mutein, other monomeric or single domain polypeptides can
be employed, including but not limited to a DARPin, a Fynomer, and
a Kunitz domain peptide.
[0030] In another aspect, the disclosure provides a CAR that
secretes a polypeptide, which may or may not define a naturally
occurring monomeric motif (e.g. a lipocalin mutein) specific to a
predetermined antigen (e.g. an immunomodulatory target such as an
immune checkpoint or costimulatory molecule) that is transduced
into a T cell for secretion thereby, bringing an added therapeutic
benefit. In this vein, the secreted polypeptide preferably is
monomeric; further, the secreted polypeptide preferably is specific
for an immunomodulatory target.
[0031] This construct can be based on T-cells with a defined
specificity (either without genetic manipulation or by transduction
with a CAR or a recombinant TCR) that are equipped with the
capacity to secrete therapeutically relevant biologics. In contrast
to TRUCK-like approaches, which are based on the secretion of
proinflammatory cytokines, the present disclosure provides for a T
cell that secretes a specific binding polypeptide that can be
antagonistic or agonistic vis-h-vis its target. In the context of
oncology indications, tumor-specific T cells that secrete biologics
that are antagonistic inhibitors of the interaction of
co-inhibitory immune receptors with their ligands (also known as
"checkpoint inhibitors") may be employed. Specific, non-limiting
target examples include PD-1 and CTLA-4. Alternatively, constructs
that allow the agonistic activation of stimulatory and/or
costimulatory immune receptors can be utilized, with examples for
the respective targets being CD3 and CD137. Further, antagonists to
anti-inflammatory cytokines such as IL-10 or TGF-also can be
employed. This approach can be termed T-cell activation-induced
localized secretion (TAILS).
[0032] The skilled worker will appreciate that this approach can
also be based on other cell types, such as NK cells or B-cells.
Further, an analogous approach can be applied to auto-inflammatory
conditions; however, in this case antagonists to stimulatory
receptors or proinflammatory cytokines or agonists of coinhibitory
immune receptors may be applied, and vehicle cells that have no
cytotoxic capacity may be employed.
[0033] TAILS can be viewed as an extension of the CAR-T/Recombinant
TCR approaches with an improved efficiency. On the other hand,
TAILS can also be viewed as an alternative to traditional
biologics, where production of the therapeutic occurs in vivo. This
firstly provides the advantage of highly localized (ant-)agonism
with concomitant minimal unwanted systemic toxicity, in particular
if biologics with a low systemic half-life are employed, and
secondly facilitates efficient delivery of biologics to any site of
action within the human body, including tissues which would
otherwise not be reachable by biologics drugs that are systemically
applied, such as the brain. Furthermore, local delivery of biologic
therapeutics has the potential to not only have a more benign
toxicity profile, but also to display an improved efficacy as it is
well known that local and systemic effects of agonists or
antagonists can be profoundly different.
[0034] The skilled worker will appreciate that the following
elements are to be considered when designing the components of a
TAILS system according to the foregoing principles: [0035] (i) The
biologic used as a secreted antagonist or agonist [0036] (ii) The
target of the CAR or (recombinant) TCR [0037] (iii) The target of
the secreted biologic [0038] (iv) The cells used as vehicles and
producers of the single-chain biologic [0039] (v) The method used
to transduce the vehicle cells [0040] (vi) In-vitro and in-vivo
assays to test the functionality of the TAILS system
[0041] Several designs are possible for the biologic used as a
secreted (ant-)agonist, with the following to be regarded as a
non-limiting example. In theory, both TRUCK-like or TAILS
approaches can be implemented using proteins consisting of multiple
subunits (i.e. multiple polypeptide chains); for example, a
heterodimeric cytokine such as IL-12 consisting of the IL-12p40 and
the IL-12p35 subunits, or an IgG antibody consisting of two heavy
chains and two light chains, as such multimeric proteins might be
assembled within a vehicle cell that is transduced with all the
subunits endcoded on suitable vectors. However, a complex
multimeric protein may incorrectly inefficiently assemble, leading
either to secretion of an undesired product with concomitant
undesired effects (for example an IL-12p40 dimer), or insufficient
generation and secretion of the multimer (for example an antibody).
Therefore, biologics used for TAILS should preferably define a
monomer, with examples including but not limited to Affibody
molecules, Affilins, Affimers, Affitins, Anticalins, Avimers,
DARPins, Fynomers, Kunitz domain peptides, Monobodies or
single-chain variants of Antibodies such as scFv and their
derivatives, camelid single-chain Antibodies, nanobodies or domain
Antibodies.
[0042] The targets recognized by the T-cell, either by the
physiological (T-cell) receptor, a CAR or recombinant TCR, include,
but are not limited to the targets described above. Here, it is of
note that targets that are tumor-associated but not necessarily
tumor cell-associated (such as VEGF-A, Ang2 and others) may be
particularly suited to provide local release of a checkpoint
inhibitor or an immune receptor agonist without inducing cell
killing of specific cells. If a CAR is employed in the approach,
the overall design will follow the principles described herein for
the Anticalin-based CAR.
[0043] Potential targets of the secreted biologic include
coinhibitory receptors or their ligands such as CTLA-4, B7-1, PD-1,
PD-L1, LAG3, BTLA, TIM3, TIGIT, CD160 or LAIR1. (Chen et al. 2013)
Here, the secreted biologic must be capable of interfering with the
ligand/receptor interaction or must otherwise block downstream
signaling. Alternatively, constructs that allow the agonistic
activation of stimulatory and/or costimulatory immune receptors can
be utilized, with examples for receptors being the TCR (e.g. via
CD3), CD28, ICOS, CD137, Ox40, GITR, HVEM, CD27, CD30, DR3, SLAM,
CD2 or CD226. (Chen et al. 2013) Depending on the target, such
agonists can for example be obtained by dimerisation or
multimerisation of a single receptor binder.
[0044] For this purpose, mobile cells that have the capacity to
strongly proliferate upon activation, and that are capable of
secreting sufficient amounts of a given biologic are required. T-,
B- and NK cells, and, where applicable, their subtypes are in
principle amenable to this task. How this would be done in practice
is well known in the art for T-cells (cf. CAR-T approach), and
could analogously be implemented for the other cell types.
[0045] Methods for cell transduction of a CAR or TCR can be
followed as described herein. Vectors suitable for T-cell
activation driven secretion are described in the literature, cf.
e.g. Chmielewski et al. 2011b; Zhang et al. 2011; Pegram et al.
2012 and Pegram et al. 2014.
[0046] The assays used to characterize engineered lymphocytes
capable of TAILS can be essentially the same as the ones described
for CAR- or TCR-transduced T-cells above. Additionally, assays can
be used to measure successful secretion of the therapeutic produced
by the cells, with ELISA assays being a straightforward
possibility.
[0047] A representative example, below, is based on a CAR-T cell
generated by transduction of T-cells with the construct
GPC3-CD137-CD3zeta (SEQ ID NO: 11) described herein. The T-cell may
be co-transduced with a suitable vector encoding the secreted
biologic; for this purpose, an Anticalin-based CTLA-4 antagonist
(SEQ ID NO: 3), which binds both human and mouse CTLA-4, can be
employed.
[0048] Also provided by the present invention is a pharmaceutical
composition comprising a T cell as described herein and optionally
a pharmaceutically acceptable carrier.
[0049] Furthermore, the present invention provides a T cell as
described herein for use in a method of treatment of cancer.
[0050] Similarly, the present invention provides a method of
treatment of cancer comprising administering to a subject in need
thereof a pharmaceutically efficient amount or numbers of T cells
as described herein.
[0051] The treatment of cancer includes the treatment of a tumor.
The subject is preferably a mammal, more preferably a human. A
pharmaceutically acceptable carrier also includes a
pharmaceutically acceptable excipient.
FIGURES
[0052] FIG. 1: Inducible expression of CTLA-4 antagonist in primary
T cells analyzed by FACS. Human peripheral blood T cells were
transduced with pSIN-(NFAT)6-.alpha.CTLA-4 and incubated for 48 h
at 37.degree. C. under activatory conditions by utilizing plates
precoated with anti-CD3 mAb and anti-CD28 mAb. Cells were stained
extracellularly using a FITC-labeled anti-CD3 antibody, and
intracellulary--after fixation and permeabilization--for SEQ ID NO:
28 utilizing a primary polyclonal anti-NGAL rabbit antibody,
followed by secondary staining with a Dylight594-conjugated
anti-rabbit IgG antibody. Immunofluorescence was analyzed using a
FACS instrument. A detailed protocol of the experiment is provided
in Example 1.
[0053] FIG. 2: Detection of functional T-cell secreted CTLA-4
antagonist by ELISA. The supernatant of human peripheral blood T
cells, mock-transduced or transduced with
pSIN-(NFAT)6-.alpha.CTLA-4 and cultured under activatory conditions
(cf. Example 1 and FIG. 1) was analyzed for the presence of
functionally active CTLA-4 antagonist SEQ ID NO: 28 by ELISA.
Recombinant human CTLA-4 was coated on an ELISA plate as described
in Example 1, and culture supernatants of transduced and
mock-transduced T cells were added. Plate-bound SEQ ID NO: 28 was
detected by primary staining with an anti-NGAL polyclonal antibody
from the rabbit, secondary staining using goat anti rabbit IgG-HRP
and addition of a chromogenic HRP substrate as described in Example
1.
[0054] FIG. 3: Detection of functional T-cell secreted CTLA-4
antagonist by FACS of CTLA-4-positive CHO cells. The supernatant of
Jurkat cells transduced with pSIN-(NFAT)6-.alpha.CTLA-4 and
cultured under activatory conditions (cf. Example 2) was analyzed
for the presence of functionally active CTLA-4 antagonist SEQ ID
NO: 28 by incubation of the supernatant with CHO cells stably
expressing CTLA-4 on their surface (CHO::CTLA-4). As a control,
CHO::CTLA-4 were incubated with isotype control. Cell-bound SEQ ID
NO: 28 that had been excreted by the Jurkat cells was then detected
using anti-NGAL polyclonal antibody followed by secondary staining
with a fluorescently labeled anti-rabbit Antibody as described in
Example 2.
[0055] FIG. 4: Inducible expression of Fc fusion of CTLA-4
antagonist in primary T cells analyzed by FACS. Human peripheral
blood T cells were transduced with pSIN-(NFAT)6-.alpha.CTLA-4-Fc
and incubated for 48 h at 37.degree. C. under activatory conditions
by utilizing plates precoated with anti-CD3 mAb and anti-CD28 mAb.
Cells were stained extracellularly using a FITC-labeled anti-CD3
antibody, and intracellulary--after fixation and
permeabilization--for SEQ ID NO: 29 utilizing a primary polyclonal
anti-NGAL antibody, followed by secondary staining with a
Dylight594-conjugated anti-rabbit IgG antibody. Immunofluorescence
was analyzed using a FACS instrument. Cf. Example 3 for
details.
[0056] FIG. 5: Detection of functional T-cell secreted Fc fusion of
a CTLA-4 antagonist by ELISA. The supernatant of human peripheral
blood T cells, mock-transduced or transduced with
pSIN-(NFAT)6-.alpha.CTLA-4-Fc and cultured under activatory
conditions (cf. Example 3 and FIG. 4) was analyzed for the presence
of the functionally active Fc fusion of the CTLA-4 antagonist SEQ
ID NO: 29 by ELISA. Recombinant human CTLA-4 was coated on an ELISA
Plate as described in Example 3, and culture supernatants of
transduced and mock-transduced T cells were added. Plate-bound SEQ
ID NO: 29 was detected by primary staining with an anti-NGAL
polyclonal rabbit antibody, secondary staining using goat anti
rabbit IgG-HRP and addition of a chromogenic HRP substrate as
described in Example 3.
WORKING EXAMPLES
[0057] A. CAR-Transduced T Cell Containing an Anticalin as CAR
Moiety
[0058] Generation of CAR-Transduced T-Cells
[0059] As a first step, the coding DNA corresponding to the SEQ ID
NOs: 8-11 is generated by gene synthesis. For that purpose, a set
of codons that is compatible with mammalian expression needs to be
employed. The DNA stretches encoding the CARs are equipped with
suitable restriction sites at the N- and C-termini to allow cloning
into the retroviral expression vector pBullet (Willemsen et al.
2000), or, alternatively, pSTITCH (Weijtens et al. 1998). The gene
synthesis is straightforwardly performed using a commercial
provider, and the resulting DNA is cloned into pBullet using
standard molecular biology methods.
[0060] To obtain retroviral particles suitable for peripheral blood
T-cell transduction we proceed as described (Weijtens et al. 1998).
In short, DNA of the respective retroviral vector (pBullet or
pSTITCH) containing the Anticalin-based CAR (6 mg DNA) is
co-transfected into 293T cells by calcium phosphate
co-precipitation with the retroviral helper plasmid DNA pHIT60 and
pCOLT (each 6 mg DNA) encoding the MuLV gag and pol genes (pHIT60)
and the GALV envelope gene (pCOLT), respectively, under control of
the CMV promotor/enhancer (Weijtens et al. 1998). This procedure
results in transient production of high titers of infectious
retrovirus. Peripheral blood lymphocytes from healthy donors are
isolated by density centrifugation and cultured for 48 h in
RPMI-1640 medium supplemented with 10% FCS in the presence of IL-2
(400 U/ml) and OKT3 MAb (100 ng/ml). Cells are harvested, washed,
resuspended in medium with IL-2 (400 U/ml), and co-cultured for 48
h with 293T cells that are transiently transfected as described
above and therefore produce and excrete the retroviral transducing
particles into the medium. In the final step, T cells are harvested
and subsequently tested for successful CAR expression and
functional assays as described further below.
[0061] Characterisation of CAR-Transduced T-Cells--FACS
[0062] Successful cell-surface expression of the chimeric antigen
receptor on transduced T-cells is demonstrated by multicolor
immunofluorescence, using the fluorescence-labeled, soluble
extracellular domains of the respective targets human c-Met and
human GPC-3, or alternatively, an anti-CD8 antibody detected by a
suitable fluorescence-labeled secondary antibody.
Immunofluorescence is analyzed using a suitable FACS instrument.
Transduced cells labeled in this manner show a clear fluorescence
signal in FACS compared to a non-transduced T-cell control.
[0063] Characterisation of CAR-Transduced T-Cells--T-Cell
Activation on Coated Plates
[0064] To investigate whether signaling via the CAR leads to T-cell
activation, we can conduct two experiments with coated protein to
cluster the CAR on the transduced T-cells. This is either
accomplished by plastic-coating an anti-CD8 antibody on a
microtiter plate or by coating the target protein of the CAR
(either c-Met or GPC-3 depending on the construct). A non-binding
IgG1 Antibody serves as the negative control. The microtiter plates
are coated with 1-50 .mu.g/ml of the anti-CD8 antibody, c-Met,
GPC-3 or control IgG1. Transduced and non-transduced peripheral
blood T cells (1-3 10.sup.5 cells/well) are incubated for 48 h at
37.degree. C. in the coated microtiter plates. After 48 h, culture
supernatants are analyzed for IFN-g by ELISA using a suitable
commercial kit.
[0065] The supernatant of CAR-expressing T-cells incubated in
plastic well coated with either an anti-CD8-antibody or with the
targets c-Met or GPC3, respectively, contains significantly more
IFN-gamma than that of the controls. The latter are either T-cells
not expressing a CAR, or CAR-transduced T-cells incubated in wells
coated with the control IgG1.
[0066] Characterisation of CAR-Transduced T-Cells--T-Cell
Activation by Coculture with Target-Expressing Mammalian Cells
[0067] In a second step, which is more akin to the desired
situation in-vivo, different amounts of receptor-grafted T cells
(1.25 to 10.times.10.sup.4 cells/well) are co-cultured for 48 h
with GPC-3+ or c-Met+ immortal tumor cell lines. Target-positive
cell lines that can be used are HepG2 (GPC-3) and H441 (c-Met), and
examples for GPC-3/c-Met-negative control cells are SK-HEP-1
(GPC-3) and A2870 (c-Met). As a further control for T-cell CAR
specificity, we co-incubate non-transduced T-cells with GPC-3+ or
c-Met+ cells. In all cases, cells are coincubated for 48 h, and
supernatants are subsequently analysed for IFN-gamma
expression.
[0068] We find that the supernatant of CAR-transduced T-cells
cocultured with the target-matched immortal cell lines contains
significantly more IFN-gamma than the controls, clearly showing
CAR-specific T-cell activation.
[0069] Characterisation of CAR-Transduced T-Cells--Measurement of
Cytotoxicity of T-Cells Cocultured with Target-Expressing Mammalian
Cells
[0070] Specific cytotoxicity of receptor-grafted T cells to target
cells is measured by lactate dehydrogenase (LDH) release. Briefly,
receptor-grafted and non-transduced T cells (1.25-10.times.10.sup.4
cells/well) are co-cultured for 12 hr with 1-3.times.10.sup.4
cells/well of either GPC3+, c-Met+, GPC3- or c-Met-cells,
respectively, in round-bottomed microtiter plates.
Effector-to-target ratios range from 10 to 1. To determine
spontaneous, baseline LDH release, T cells are cultured without
target cells. LDH in culture supernatants (100 ml/well) is
determined utilizing a suitable commercial kit. The percentage of
specific cytotoxicity is calculated by correcting for spontaneous
release and normalizing to the maximum LDH release determined after
addition of 1% (v/v) Nonidet P-40.
[0071] We find that the specific cytotoxicity of the Anticalin-CAR
based T-cells is significantly higher in the presence of the
matched, target-positive tumor cell lines than in the presence of
the target-negative tumor cell lines. Additionally, non-transduced
T-cell preparations show a much reduced specific cytotoxicity
compared to the transduced T-cells.
[0072] In-Vivo Characterization of CAR-Transduced T-Cells
[0073] The following experiment is performed in analogous fashion
to Chmielewski et al. 2011b. In brief, human T cells are isolated
from peripheral blood by magnetic activated cell sorting, using
human CD3+ beads. The transduction-competent retroviri encoding the
c-Met- and GPC3-specific CARs are generated and used for
transduction of human T-cells as described above. For the mouse
studies, we use the NIH-Ill mouse (Charles River), which is
deficient in NK cells, B cells, and T cells. MC38 cells stably
transfected with human GPC-3 or c-Met (9.times.10.sup.5
cells/mouse) are s.c. coinjected together with the engineered,
specificity matched T cells (2.times.10.sup.5 T cells/mouse) into
NIH-Ill mice (6-7 mice per group). Alternatively, tumors are
induced by s.c. injection of MC38 cells stably transfected with
human GPC-3 or c-Met and matched CAR-T cells are applied by i.v.
injection at day 6. The respective experiment using CAR-T cells and
the mismatched transfected tumor cell line serves as a negative
control. Tumor growth is monitored daily by external measurement
with a digital caliper.
[0074] In the experiments, we find a significant delay in tumor
outgrowth when target-positive cells and target-specific CAR-T
cells are injected into the same mouse compared to negative
controls.
[0075] B. CAR-Transduced T Cell Containing an Anticalin as TAILS
Moiety
[0076] Generation of CAR-Transduced T-Cells Capable of TAILS
(CAR/TAILS-T)
[0077] The construct GPC3-CD137-CD3zeta (SEQ ID NO: 11) is
generated and cloned into a suitable retroviral vector as described
above. Retroviral particles suitable for peripheral blood T-cell
transduction are obtained by co-transfection with retroviral helper
particles into 293T cells, also as described above.
[0078] A vector suitable to facilitate retroviral expression of the
T-cell activation inducible Anticalin-based CTLA4-antagonist
(".alpha.CTLA-4") is obtained by exchanging the mature IL-12
sequence in pSIN-(NFAT)6-IL-12 (Chmielewski et al. 2011b) by the
Anticalin sequence (SEQ ID NO: 3) using standard molecular biology
methods, to obtain pSIN-(NFAT)6-.alpha.CTLA-4. Note that the leader
sequence of IL-12 remains in the construct to serve as a secretion
signal.
[0079] In a 2-step transduction procedure, T cells are retrovirally
transduced with the .alpha.CTLA-4 expression cassette and
positively selected in the presence of 0.5 mg/mL Geneticin (G418)
and IL-2 (500 IU/mL) on plates precoated with anti-CD3 mAb OKT-3
and anti-CD28 mAb 15E8 as previously described (Chmielewski et al.
2011b). Geneticin-resistant clones are transduced with the
GPC-3-specific CAR GPC3-CD137-CD3zeta.
[0080] Characterization of CAR/TAILS T-Cells
[0081] Successful cell-surface expression of the chimeric antigen
receptor on the doubly transduced CAR/TAILS T-cells is performed as
described above: we employ multicolor immunofluorescence, using the
fluorescence-labeled, soluble extracellular domain of human GPC-3,
or alternatively, an anti-CD8 antibody detected by a suitable
fluorescence-labeled secondary antibody. Immunofluorescence is
analyzed using a suitable FACS instrument. Transduced cells labeled
in this manner show a clear fluorescence signal in FACS compared to
a non-transduced T-cell control.
[0082] Characterisation of CAR/TAILS-T-Cells--T-Cell Activation on
Coated Plates and CTLA-4 Secretion
[0083] To investigate whether signaling via the CAR leads to T-cell
activation in the CAR/TAILS T-cell, we conduct two experiments with
coated protein to cluster the CAR on the transduced T-cells, in
analogy to the experiment described above. This is either
accomplished by plastic-coating an anti-CD8 antibody on a
microtiter plate, or, alternatively, by coating GPC-3. A
non-binding IgG1 Antibody serves as the negative control. The
microtiter plates are coated with 1-50 .mu.g/ml of the anti-CD8
antibody, GPC-3 or control IgG1. Transduced and non-transduced
peripheral blood T cells (1-3 10.sup.5 cells/well) are incubated
for 48 h at 37.degree. C. in the coated microtiter plates. After 48
h, culture supernatants are analyzed for IFN-gamma by ELISA using a
suitable commercial kit. Further, supernatants are analyzed for the
presence of the Anticalin-based CTLA-4 antagonist by a suitable
ELISA setup.
[0084] The supernatant of CAR-expressing T-cells incubated in
plastic well coated with either an anti-CD8-antibody or with GPC3,
respectively, contains significantly more IFN-gamma and
Anticalin-based CTLA-4 antagonist than that of the controls. The
latter are either T-cells not expressing a CAR, but transduced with
.alpha.CTLA-4 only (SEQ ID NO: 3), or doubly transduced CAR/TAILS
T-cells incubated in wells coated with the control IgG1.
[0085] Characterization of CAR/TAILS-T-Cells--T-Cell Activation by
Coculture with Target-Expressing Mammalian Cells
[0086] In a second step, which is more akin to the desired
situation in-vivo, different amounts of receptor-grafted T cells
(1.25 to 10.times.10.sup.4 cells/well) are co-cultured for 48 h
with GPC-3+ immortal tumor cell lines. The GPC3-positive cell line
used is HepG2, and the GPC-3-negative control cell line is
SK-HEP-1. As a further control for T-cell CAR specificity, we
co-incubate with GPC-3+ cells with T-cells not expressing a CAR,
but transduced with .alpha.CTLA-4 (SEQ ID NO: 3) only. In all
cases, cells are coincubated for 48 h, and supernatants are
subsequently analysed for IFN-.gamma. expression and the expression
of the anti-CTLA-4 Anticalin.
[0087] We find that the supernatant of CAR-transduced T-cells
co-cultured with the target-matched immortal cell lines contains
significantly more IFN-.gamma. and anti-CTLA-4 Anticalin than the
controls, clearly showing CAR-specific T-cell activation and T-cell
activation induced secretion of the CTLA-4 antagonist.
[0088] Characterisation of CAR/TAILS-T-Cells--Measurement of
Cytotoxicity of T-Cells Cocultured with Target-Expressing Mammalian
Cells
[0089] Specific cytotoxicity of receptor-grafted T cells to target
cells is measured by lactate dehydrogenase (LDH) release as
described above. Briefly, doubly transduced CAR/TAILS T-cells,
CAR-transduced-cells or non-transduced T cells
(1.25-10.times.10.sup.4 cells/well) are co-cultured for 12 hr with
1-3.times.10.sup.4 cells/well of either GPC3+, or GPC3- cells,
respectively, in round-bottomed microtiter plates.
Effector-to-target ratios range from 10 to 1. To determine
spontaneous, baseline LDH release, T cells are cultured without
target cells. LDH in culture supernatants (100 ml/well) is
determined utilizing a suitable commercial kit. The percentage of
specific cytotoxicity is calculated by correcting for spontaneous
release and normalizing to the maximum LDH release determined after
addition of 1% (v/v) Nonidet P-40.
[0090] We find that the specific cytotoxicity of the doubly
transduced CAR/TAILS T-cells is at least retained compared to the
CAR-only based T-cells, and significantly higher in the presence of
the matched, target-positive tumor cell lines than in the presence
of the target-negative tumor cell lines. Additionally,
non-transduced T-cell preparations show a much reduced specific
cytotoxicity compared to the transduced T-cells.
[0091] Characterisation of CAR/TAILS-T-Cells--In-Vivo
Experiment
[0092] The following experiment is performed in analogy to
Chmielewski et al. 2011b and above, but utilizing a different tumor
cell line. In brief, human T cells are isolated from peripheral
blood by magnetic activated cell sorting, using human CD3+ beads.
The GPC-3-specific and .alpha.CTLA-4-Anticalin secreting
CAR/TAILS-T cells are generated as described herein. For the mouse
studies, we use the NIH-Ill mouse (Charles River), which is
deficient in NK cells, B cells, and Tcells. AT3 cells stably
transfected with human GPC-3 (9.times.10.sup.5 cells/mouse) are
s.c. coinjected together with the engineered CAR/TAILS-T cells
(2.times.10.sup.5 T cells/mouse) into NIH-Ill mice (6-7 mice per
group). Alternatively, tumors are induced by s.c. injection of AT3
cells stably transfected with human GPC-3 and CAR/TAILS-T cells are
applied by i.v. injection at day 6. Negative controls include a
vehicle injection control, the respective experiment using
CAR/TAILS-T and mock-transfected AT3 cells, and the respective
experiment utilizing CAR-T cells that do not secrete .alpha.-CTLA-4
Anticalin. Tumor growth is monitored daily by external measurement
with a digital caliper.
[0093] In the experiments, we find a significant delay in tumor
outgrowth when target-positive cells and target-specific
CAR/TAILS-T cells are injected into the same mouse compared to the
controls.
Example 1: Generation of Primary T Cells Capable of TAILS and
Characterisation
[0094] A vector suitable to facilitate retroviral expression of the
T-cell activation inducible Anticalin-based CTLA4-antagonist
(".alpha.CTLA-4") was obtained by replacing the IL-12 sequence in
pSIN-(NFAT)6-IL-12 (Chmielewski et al. 2011b) by the Anticalin
sequence (SEQ ID NO: 3) with a Strep-tag II (SEQ ID NO: 27) using
standard molecular biology methods, to obtain
pSIN-(NFAT)6-.alpha.CTLA-4. The leader sequence of the mouse
immunoglobulin kappa light chain (GenBank Acc #CAB46127.1) instead
of the IL-12 leader was employed in the construct to serve as a
secretion signal. The final amino acid sequence corresponds to SEQ
ID NO: 23, encoded by the DNA sequence SEQ ID NO: 24.
[0095] To obtain retroviral particles suitable for peripheral blood
T-cell transduction we proceeded as described (Weijtens et al.
1998; Cheadle et al., 2012). In short, DNA of
pSIN-(NFAT)6-.alpha.CTLA-4 (6 mg DNA) was co-transfected into 293T
cells by calcium phosphate co-precipitation with the retroviral
helper plasmid DNA pHIT60 and pCOLT (each 6 mg DNA) encoding the
MuLV gag and pol genes (pHIT60) and the GALV envelope gene (pCOLT),
respectively, under control of the CMV promotor/enhancer (Weijtens
et al. 1998). This procedure resulted in the production of high
titers of infectious retrovirus.
[0096] In order to obtain T cells transduced with
pSIN-(NFAT)6-.alpha.CTLA-4, peripheral blood lymphocytes from
healthy donors were isolated by density centrifugation in a
Ficoll-Paque (GE Healthcare) density gradient and cultured for 48 h
in RPM11640 medium supplemented with 10% (v/v) FCS in the presence
of IL-2 (400 U/ml) and OKT3 mAb (100 ng/ml). Cells were harvested,
washed, resuspended in medium with IL-2 (400 U/ml), and co-cultured
for 48 h with 293T cells that were transiently transfected as
described above and therefore released the retrovirus particles
into the medium. In the final step, T cells were harvested. The
same procedure was also carried out using mock-transduction, i.e.
transducing the gag, pol and env genes of the retrovirus without
the expression cassette.
[0097] To investigate whether activation of the transduced T cells
induced the secretion of SEQ ID NO: 28 (the mature processed form
of SEQ ID NO:24), 5.times.10.sup.6 cells peripheral blood T cells
in 10 mL medium--transduced or mock-transduced--were incubated for
48 h at 37.degree. C. on Nunc.TM. OmniTray.TM. plates precoated
with anti-CD3 mAb OKT-3 and anti-CD28 mAb 15E8 as previously
described (Chmielewski et al. 2011b). To determine both successful
expression of the anti-CTLA-4 Anticalin (SEQ ID NO: 28) on the
transduced TAILS T-cells and the fraction of cells that was
successfully transduced, we employed multicolor immunofluorescence
using a FITC-labeled anti-CD3 antibody (anti-human CD3, clone OKT3
(ATCC)), and a polyclonal anti-NGAL rabbit antibody, followed by
secondary staining with an anti-rabbit antibody labeled with the
fluorescent dye Dylight594. The detailed protocol was as follows,
performing all incubation steps protected from light: Transduced T
cells were stained for CD3 using the FITC-labeled anti-CD3 antibody
for 30 minutes at 4.degree. C., followed by fixation and
permeabilization by resuspending in 250 .mu.l BD Cytofix/Cytoperm
solution and incubating for 20 minutes at 4.degree. C., followed by
washing twice in a buffer containing the cell permeabilizing agent
saponin (BD Perm/Wash.TM. buffer, Cat. 554723). Intracellular
staining of SEQ ID NO: 28 was then achieved by thoroughly
resuspending the fixed and permeabilized cells in 50 .mu.L of a
saponin-containing buffer (BD Perm/Wash.TM. buffer) containing 0.5
.mu.g anti-NGAL mAb per 1 million cells, washing twice with
saponin-containing BD Perm/Wash.TM. buffer, and incubation for 30
minutes at 4.degree. C. with 1 .mu.g of the Dylight594-conjugated
anti-rabbit IgG antibody per 1 million cells. Finally, cells were
washed twice with PBS buffer and immunofluorescence was analyzed
using a BD FACS Canto II instrument. In the FACS analysis,
transduced cells labeled in this manner showed a clearly positive
anti-NGAL reactivity (FIG. 1A) compared to the mock-transduced
T-cell control (FIG. 1B). We found that according to a threshold
set based on the mock-transduced T-cell control (FIG. 1B), 52% of
primary T cells had been transduced and were found to be SEQ ID NO:
28-positive.
[0098] Supernatants were analyzed for the presence of functionally
active Anticalin-based CTLA-4 antagonist by ELISA as described in
the following: Recombinant human CTLA-4 at a concentration of 5
.mu.g/mL in PBS was added to each well of a 384 well ELISA plate
and incubated over night at 4.degree. C. All following steps were
performed with 1 h incubation time and repeated washing with
PBS/0.05% (w/v) Tween20 (PBS-T). In the first step, plates were
blocked (BSA 2% (w/v) in PBS-T/0.1% (w/v) Tween 20) and 20 .mu.L of
culture supernatants were added. Subsequently, rabbit anti-NGAL
polyclonal antibody at a concentration of 1 .mu.g/mL in PBS/0.1%
(w/v) Tween20/2% (w/v) BSA was added, and bound antibody was
detected using goat anti-rabbit IgG-HRP in a dilution of 1:5,000 in
PBS/0.1% (w/v) Tween20/2% (w/v) BSA. Chromogenic 3,3',
5,5;-tetramethylbenzidine (TMB) substrate was used as a detection
agent according to the manufacturer's instructions (Life
Technologies). Fluorescence signals in RFU (relative fluorescence
units) were measured using a plate fluorescence reader.
[0099] We found a clear anti-CTLA-4 reactivity in supernatants of
pSIN-(NFAT)6-.alpha.CTLA-4-transduced and activated T cells
compared to the supernatants of mock-transfected cells (FIG. 2).
Note that the mock-transfected cells showed a background
fluorescence signal due to nonspecific matrix effects, which was,
however, significantly below the positive ELISA signal of the
supernatant of pSIN-(NFAT)6-.alpha.CTLA-4-transduced cells.
Example 2: Target Cell Binding of TAILS-Produced Anti-CTLA-4
Anticalin
[0100] The experiment of this example was carried out in analogy to
Example 1, but using the Jurkat T cell line instead of primary T
cells. In short, Jurkat T cells were transduced with
pSIN-(NFAT)6-.alpha.CTLA-4 as described in Example 1, and incubated
in medium on plates precoated with anti-CD3 mAb OKT-3 and anti-CD28
mAb 15E8 as previously described (Chmielewski et al. 2011b).
[0101] The culture supernatant containing the anti-CTLA-4 Anticalin
(SEQ ID NO: 28) was obtained from the Jurkat cells by
centrifugation and added to Chinese Hamster Ovary cells stably
transfected with CTLA-4 (CHO::CTLA-4). As a control, CHO::CTLA-4
were incubated with an isotype control antibody. Cell-bound SEQ ID
NO: 28 was detected using anti-NGAL rabbit polyclonal antibody
followed by staining with anti-rabbit antibody labeled with the
fluorescent dye Dylight594. In the FACS fluorescence histogram
(FIG. 3), a clear shift in fluorescence intensity for cells
incubated with the supernatant from
pSIN-(NFAT)6-.alpha.CTLA-4-transduced Jurkat cells compared to the
isotype control is evident, showing the binding of excreted SEQ ID
NO: 28 to CTLA-4 positive cells.
Example 3: Generation of Primary T Cells Capable of Inducible
Secretion of an Fc-Fusion of Anticalin-Based CTLA-4 Antagonist and
Characterisation
[0102] The experiments described here were performed in full
analogy to Example 1, but utilizing SEQ ID NO: 25, which is a
fusion of the Anticalin-based CTLA-4 Antagonist (SEQ ID NO: 23) to
the Fc fragment of a human IgG1 antibody. The DNA sequence encoding
SEQ ID NO: 25 is provided by SEQ ID NO: 26. The corresponding
inducible expression vector pSIN-(NFAT)6-.alpha.CTLA-4-Fc was
generated by standard molecular biology methods.
[0103] Generation of retroviral particles suitable for peripheral
blood T-cell transduction, as well as T cell preparation and
transduction with pSIN-(NFAT)6-.alpha.CTLA-4-Fc, were performed as
described in Example 1. Mock-transduction was used as a control. To
investigate whether activation of the transduced T cells lead to
secretion of SEQ ID NO: 29 (the mature processed form of SEQ ID
NO:25), we proceeded with multicolor FACS staining of the targets
CD3 and SEQ ID NO: 29 as described in Example 1. In the FACS
analysis, transduced and fluorescence-labeled cells showed a
clearly positive anti-NGAL reactivity (FIG. 4A) compared to the
mock-transduced T-cell control (FIG. 4B). We found that according
to a threshold set based on the mock-transduced T-cell control
(FIG. 4B), 63% of primaryT cells had been transduced and were found
to be SEQ ID NO: 29-positive.
[0104] Supernatants were analyzed for the presence of functionally
active Anticalin-based CTLA-4 antagonist .alpha.CTLA-4-Fc (SEQ ID
NO: 29) by ELISA as described in Example 1. In the ELISA, we found
a clear anti-CTLA-4 reactivity for T cells transduced with
pSIN-(NFAT)6-.alpha.CTLA-4-Fc and cultured under activatory
conditions, compared to the mock-transfected cells (FIG. 5). Note
that the mock-transfected cells showed a background fluorescence
signal due to nonspecific matrix effects, which was, however,
significantly below the positive ELISA signal of the supernatant of
pSIN-(NFAT)6-.alpha.CTLA-4-Fc-transduced cells.
TABLE-US-00001 TABLE 1 Amino acid SEQ ID NO's in this application
and corresponding SEQ ID NO Amino acid sequence DNA sequence SEQ ID
NO: 1 SEQ ID NO: 12 SEQ ID NO: 2: SEQ ID NO: 13 SEQ ID NO: 3 SEQ ID
NO: 14 SEQ ID NO: 4: SEQ ID NO: 15 SEQ ID NO: 5 SEQ ID NO: 16 SEQ
ID NO: 6 SEQ ID NO: 17 SEQ ID NO: 7 SEQ ID NO: 18 SEQ ID NO: 8 SEQ
ID NO: 19 SEQ ID NO: 9 SEQ ID NO: 20 SEQ ID NO: 10 SEQ ID NO: 21
SEQ ID NO: 11 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO:
25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID
NO: 30
REFERENCES
[0105] Birkholz, K., et al. (2009). "Transfer of mRNA encoding
recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for
use in the adoptive immunotherapy of cancer." Gene Ther 16(5):
596-604. [0106] Brocks, B., et al. (1997). "A TNF receptor
antagonistic scFv, which is not secreted in mammalian cells, is
expressed as a soluble mono- and bivalent scFv derivative in insect
cells." Immunotechnology 3(3): 173-84. [0107] Cheadle, E J., et al.
(2012). "Chimeric antigen receptors for T-cell based therapy."
Chapter 36, in: "Antibody engineering: methods and protocols", 2nd
Edition, Ed. P. Chames, Meth. Mol. Biol. 907, 645-666. [0108] Chen,
L., et al. (2013). "Molecular mechanisms of T cell co-stimulation
and co-inhibition." Nat Rev Immunol 13(4): 227-42. [0109]
Chmielewski, M., et al. (2012). "T cells that target
carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas
without inducing autoimmune colitis in mice." Gastroenterology
143(4): 1095-107 e2. [0110] Chmielewski, M., et al. (2011)a. "CD28
signalling does not affect the activation threshold in a chimeric
antigen receptor-redirected T-cell attack." Gene Ther 18(1): 62-72.
[0111] Chmielewski, M., et al. (2013)a. "Antigen-Specific T-Cell
Activation Independently of the MHC: Chimeric Antigen
Receptor-Redirected T Cells." Front Immunol 4: 371. [0112]
Chmielewski, M., et al. (2014). "Of CARs and TRUCKs: chimeric
antigen receptor (CAR) T cells engineered with an inducible
cytokine to modulate the tumor stroma." Immunol Rev 257(1): 83.90.
[0113] Chmielewski, M., et al. (2011)b. "IL-12 release by
engineered T cells expressing chimeric antigen receptors can
effectively Muster an antigen-independent macrophage response on
tumor cells that have shut down tumor antigen expression." Cancer
Res 71(17): 5697-706. [0114] Chmielewski, M., et al. (2013)b. "T
cells redirected by a CD3zeta chimeric antigen receptor can
establish self-antigen-specific tumour protection in the long
term." Gene Ther 20(2): 177-86. [0115] Ewert, S., et al. (2003)a.
"Structure-based improvement of the biophysical properties of
immunoglobulin VH domains with a generalizable approach."
Biochemistry 42(6): 1517-28. [0116] Ewert, S., et al. (2003)b.
"Biophysical properties of human antibody variable domains." J Mol
Biol 325(3): 531-53. [0117] Haisma, H. J., et al. (1998)a.
"Construction and characterization of a fusion protein of
single-chain anti-carcinoma antibody 323/A3 and human
beta-glucuronidase." Cancer Immunol Immunother 45(5): 266-72.
[0118] Haisma, H. J., et al. (1998)b. "Construction and
characterization of a fusion protein of single-chain anti-CD20
antibody and human beta-glucuronidase for antibody-directed enzyme
prodrug therapy." Blood 92(1): 184-90. [0119] Hombach, A., et al.
(2010). "Adoptive immunotherapy with genetically engineered T
cells: modification of the IgG1 Fc `spacer` domain in the
extracellular moiety of chimeric antigen receptors avoids
`off-target` activation and unintended initiation of an innate
immune response." Gene Ther 17(10): 1206-13. [0120] Hombach, A. A.,
et al. (2013). "Arming cytokine-induced killer cells with chimeric
antigen receptors: CD28 outperforms combined CD28-OX40
"super-stimulation"." Mol Ther 21(12): 2268-77. [0121] Jaalouk, D.
E., et al. (2006). "A self-inactivating retrovector incorporating
the IL-2 promoter for activation-induced transgene expression in
genetically engineered T-cells." Virol J 3: 97. [0122] Kershaw, M.
H., et al. (2006). "A phase I study on adoptive immunotherapy using
gene-modified T cells for ovarian cancer." Clin Cancer Res 12(20 Pt
1): 6106-15. [0123] Kofler, D. M., et al. (2011). "CD28
costimulation impairs the efficacy of a redirected t-cell antitumor
attack in the presence of regulatory t cells which can be overcome
by preventing Lck activation." Mol Ther 19(4): 760-7. [0124] Maiti,
S. N., et al. (2013). "Sleeping beauty system to redirect T-cell
specificity for human applications." J Immunother 36(2): 112-23.
[0125] Maliar, A., et al. (2012). "Redirected T cells that target
pancreatic adenocarcinoma antigens eliminate tumors and metastases
in mice." Gastroenterology 143(5): 1375-84 e1-5. [0126] Maude, S.
L., et al. (2014). "Chimeric antigen receptor T cells for sustained
remissions in leukemia." N Engl J Med 371(16): 1507-17. [0127]
Pegram, H. J., et al. (2012). "Tumor-targeted T cells modified to
secrete IL-12 eradicate systemic tumors without need for prior
conditioning." Blood 119(18): 4133-41. [0128] Pegram, H. J., et al.
(2014). "IL-12-secreting CD19-targeted cord blood-derived T cells
for the immunotherapy of B-cell acute lymphoblastic leukemia."
Leukemia. [0129] Peipp, M., et al. (2004). "Efficient eukaryotic
expression of fluorescent scFv fusion proteins directed against CD
antigens for FACS applications." J Immunol Methods 285(2): 265-80.
[0130] Rosenberg, S. A., et al. (1990). "Gene transfer into
humans--immunotherapy of patients with advanced melanoma, using
tumor-infiltrating lymphocytes modified by retroviral gene
transduction." N Engl J Med 323(9): 570-8. [0131] Schaefer, J. V.,
et al. (2012). "Transfer of engineered biophysical properties
between different antibody formats and expression systems." Protein
Eng Des Sel 25(10): 485-506. [0132] Shi, H., et al. (2014).
"Chimeric antigen receptor for adoptive immunotherapy of cancer:
latest research and future prospects." Mol Cancer 13: 219. [0133]
Textor, A., et al. (2014). "Efficacy of CAR T-cell Therapy in Large
Tumors Relies upon Stromal Targeting by IFNgamma." Cancer Res
74(23): 6796-805. [0134] Weijtens, M. E., et al. (1998). "A
retroviral vector system `STITCH` in combination with an optimized
single chain antibody chimeric receptor gene structure allows
efficient gene transduction and expression in human T lymphocytes."
Gene Ther 5(9): 1195-203. [0135] Willemsen, R. A., et al. (2000).
"Grafting primary human T lymphocytes with cancer-specific chimeric
single chain and two chain TCR." Gene Ther 7(16): 1369.77. [0136]
Zhang, L., et al. (2011). "Improving adoptive T cell therapy by
targeting and controlling IL-12 expression to the tumor
environment." Mol Ther 19(4): 751-9. [0137] Zhao, Y., et al.
(2006). "Transduction of an HLA-DP4-restricted NY-ESO-1-specific
TCR into primary human CD4+ lymphocytes." J Immunother 29(4):
398-406.
Sequence CWU 1
1
301152PRTartificialLipocalin mutein binding c-Met 1Ala Ser Asp Glu
Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala Met Thr
Val Asp Thr Gln Asp Pro Leu Ser Leu Tyr Val Ser Val 20 25 30Ser Pro
Ile Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Thr 35 40 45Val
Thr Leu Asn Gln Ile Gly Arg Ser Gln Glu Val Leu Ala Val Leu 50 55
60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Leu Tyr Gly Gly Ala His65
70 75 80Val Ala Tyr Ile Gln Arg Ser His Val Lys Asp His Tyr Ile Phe
Tyr 85 90 95Ser Glu Gly Asp Thr Trp Gly Gly Pro Val Pro Gly Val Trp
Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu
Asp Phe Glu Lys 115 120 125Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu
Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu Thr Ser Ser Pro
Gly145 1502178PRTartificialLipocalin mutein binding GPC-3 2Gln Asp
Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro
Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25
30Val Val Gly Arg Ala Gly Asn Val Ala Leu Arg Glu Asp Lys Asp Pro
35 40 45Pro Lys Met Arg Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser
Tyr 50 55 60Asn Val Thr Asn Val Arg Phe Ala Met Lys Lys Cys Met Tyr
Ser Ile65 70 75 80Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe
Thr Leu Gly Gln 85 90 95Ile Lys Ser Glu Pro Gly Asn Thr Ser Asn Leu
Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met Val
Phe Phe Lys Glu Val Tyr Gln 115 120 125Asn Arg Glu Ile Phe Phe Ile
Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys
Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro
Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175Asp Gly3178PRTartificialLipocalin mutein binding CTLA-4 3Gln Asp
Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro
Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25
30Val Val Gly Leu Ala Gly Asn Arg Ile Leu Arg Gln Asp Gln His Pro
35 40 45Met Leu Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser
Tyr 50 55 60Gln Val Thr Ser Val Ile Ser Ser His Lys Lys Cys Leu Tyr
Pro Ile65 70 75 80Ala Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe
Thr Leu Gly Asn 85 90 95Ile Lys Ser Tyr Gly Asp Lys Val Ser Tyr Leu
Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met Val
Phe Phe Lys His Ala Asp Thr 115 120 125Asn Tyr Glu Ser Phe Ser Ile
Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys
Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro
Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175Asp Gly469PRTartificialCD8 linker and transmembrane region 4Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala1 5 10
15Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
Ile 35 40 45Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
Leu Val 50 55 60Ile Thr Leu Tyr Cys655113PRTartificialintracellular
signaling domain of CD3zeta 5Leu Arg Val Lys Phe Ser Arg Ser Ala
Asp Ala Pro Ala Tyr Lys Gln1 5 10 15Gly Gln Asn Gln Leu Tyr Asn Glu
Leu Asn Leu Gly Arg Arg Glu Glu 20 25 30Tyr Asp Val Leu Asp Lys Arg
Arg Gly Arg Asp Pro Glu Met Gly Gly 35 40 45Lys Pro Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu65 70 75 80Arg Arg Arg
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95Ala Thr
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105
110Arg644PRTartificialintracellular signaling domain of CD28 6Phe
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met1 5 10
15Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
20 25 30Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35
40741PRTartificialintracellular signaling domain of CD137 7Lys Arg
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25
30Pro Glu Glu Glu Glu Gly Gly Cys Glu 35
408378PRTartificialc-Met-CD28-CD3zeta polypeptide 8Ala Ser Asp Glu
Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala Met Thr
Val Asp Thr Gln Asp Pro Leu Ser Leu Tyr Val Ser Val 20 25 30Ser Pro
Ile Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Thr 35 40 45Val
Thr Leu Asn Gln Ile Gly Arg Ser Gln Glu Val Leu Ala Val Leu 50 55
60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Leu Tyr Gly Gly Ala His65
70 75 80Val Ala Tyr Ile Gln Arg Ser His Val Lys Asp His Tyr Ile Phe
Tyr 85 90 95Ser Glu Gly Asp Thr Trp Gly Gly Pro Val Pro Gly Val Trp
Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu
Asp Phe Glu Lys 115 120 125Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu
Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu Thr Ser Ser Pro Gly
Thr Thr Thr Pro Ala Pro Arg Pro145 150 155 160Pro Thr Pro Ala Pro
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 165 170 175Glu Ala Cys
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 180 185 190Asp
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 195 200
205Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Phe Trp Val
210 215 220Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
Met Thr225 230 235 240Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
Gln Pro Tyr Ala Pro 245 250 255Pro Arg Asp Phe Ala Ala Tyr Arg Ser
Leu Arg Val Lys Phe Ser Arg 260 265 270Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr Asn 275 280 285Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 290 295 300Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro305 310 315
320Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
325 330 335Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 340 345 350Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp 355 360 365Ala Leu His Met Gln Ala Leu Pro Pro Arg
370 3759375PRTartificialc-Met-CD137-CD3zeta polypeptide 9Ala Ser
Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala
Met Thr Val Asp Thr Gln Asp Pro Leu Ser Leu Tyr Val Ser Val 20 25
30Ser Pro Ile Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Thr
35 40 45Val Thr Leu Asn Gln Ile Gly Arg Ser Gln Glu Val Leu Ala Val
Leu 50 55 60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Leu Tyr Gly Gly
Ala His65 70 75 80Val Ala Tyr Ile Gln Arg Ser His Val Lys Asp His
Tyr Ile Phe Tyr 85 90 95Ser Glu Gly Asp Thr Trp Gly Gly Pro Val Pro
Gly Val Trp Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu Asp Phe Glu Lys 115 120 125Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu Thr Ser
Ser Pro Gly Thr Thr Thr Pro Ala Pro Arg Pro145 150 155 160Pro Thr
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 165 170
175Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
180 185 190Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys 195 200 205Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
Cys Lys Arg Gly 210 215 220Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe Met Arg Pro Val225 230 235 240Gln Thr Thr Gln Glu Glu Asp
Gly Cys Ser Cys Arg Phe Pro Glu Glu 245 250 255Glu Glu Gly Gly Cys
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 260 265 270Ala Pro Ala
Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 275 280 285Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 290 295
300Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
Gly305 310 315 320Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
Ala Tyr Ser Glu 325 330 335Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
Lys Gly His Asp Gly Leu 340 345 350Tyr Gln Gly Leu Ser Thr Ala Thr
Lys Asp Thr Tyr Asp Ala Leu His 355 360 365Met Gln Ala Leu Pro Pro
Arg 370 37510404PRTartificialGPC3-CD28-CD3zeta polypeptide 10Gln
Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10
15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30Val Val Gly Arg Ala Gly Asn Val Ala Leu Arg Glu Asp Lys Asp
Pro 35 40 45Pro Lys Met Arg Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys
Ser Tyr 50 55 60Asn Val Thr Asn Val Arg Phe Ala Met Lys Lys Cys Met
Tyr Ser Ile65 70 75 80Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu
Phe Thr Leu Gly Gln 85 90 95Ile Lys Ser Glu Pro Gly Asn Thr Ser Asn
Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met
Val Phe Phe Lys Glu Val Tyr Gln 115 120 125Asn Arg Glu Ile Phe Phe
Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu
Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu
Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175Asp Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
180 185 190Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
Pro Ala 195 200 205Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
Ala Cys Asp Ile 210 215 220Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
Gly Val Leu Leu Leu Ser225 230 235 240Leu Val Ile Thr Leu Tyr Cys
Phe Trp Val Arg Ser Lys Arg Ser Arg 245 250 255Leu Leu His Ser Asp
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 260 265 270Thr Arg Lys
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 275 280 285Tyr
Arg Ser Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 290 295
300Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
Arg305 310 315 320Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg Asp Pro Glu 325 330 335Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
Gln Glu Gly Leu Tyr Asn 340 345 350Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu Ile Gly Met 355 360 365Lys Gly Glu Arg Arg Arg
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 370 375 380Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala385 390 395 400Leu
Pro Pro Arg11401PRTartificialGPC3-CD137-CD3zeta polypeptide 11Gln
Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10
15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30Val Val Gly Arg Ala Gly Asn Val Ala Leu Arg Glu Asp Lys Asp
Pro 35 40 45Pro Lys Met Arg Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys
Ser Tyr 50 55 60Asn Val Thr Asn Val Arg Phe Ala Met Lys Lys Cys Met
Tyr Ser Ile65 70 75 80Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu
Phe Thr Leu Gly Gln 85 90 95Ile Lys Ser Glu Pro Gly Asn Thr Ser Asn
Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met
Val Phe Phe Lys Glu Val Tyr Gln 115 120 125Asn Arg Glu Ile Phe Phe
Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu
Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu
Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175Asp Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
180 185 190Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
Pro Ala 195 200 205Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
Ala Cys Asp Ile 210 215 220Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
Gly Val Leu Leu Leu Ser225 230 235 240Leu Val Ile Thr Leu Tyr Cys
Lys Arg Gly Arg Lys Lys Leu Leu Tyr 245 250 255Ile Phe Lys Gln Pro
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 260 265 270Asp Gly Cys
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 275 280 285Leu
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln 290 295
300Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
Glu305 310 315 320Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
Glu Met Gly Gly 325 330 335Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
Leu Tyr Asn Glu Leu Gln 340 345 350Lys Asp Lys Met Ala Glu Ala Tyr
Ser Glu Ile Gly Met Lys Gly Glu 355 360 365Arg Arg Arg Gly Lys Gly
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 370 375 380Ala Thr Lys Asp
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro385 390 395
400Arg12456DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 1 12gcttctgatg aagaaatcca ggatgtgtct
ggcacctggt acctgaaggc tatgaccgtg 60gatacccagg atccgctgtc tctgtacgtg
tctgtgtctc cgatcaccct gaccaccctg 120gaaggcggca acctggaagc
taccgtgacc ctgaaccaga tcggccgttc tcaggaagtg 180ctggctgtgc
tggaaaagac cgatgaaccg ggcaagtaca ccctgtacgg cggcgctcat
240gtggcttaca tccagcgttc tcatgtgaag gatcattaca tcttctactc
tgaaggcgat 300acctggggcg gcccggtgcc gggcgtgtgg ctggtgggcc
gtgatccgaa gaacaacctg 360gaagctctgg aagatttcga aaaggctgct
ggcgctcgtg gcctgtctac cgaatctatc 420ctgatcccgc gtcagtctga
aacctcttct ccgggc 45613534DNAartificialnucleotide sequence encoding
amino acid sequence of SEQ ID NO 2 13caggattcta cctctgatct
gatcccggct ccgccgctgt ctaaggtgcc gctgcagcag 60aacttccagg ataaccagtt
ccatggcaag tggtacgtgg tgggccgtgc tggcaacgtg 120gctctgcgtg
aagataagga tccgccgaag atgcgtgcta ccatctacga actgaaggaa
180gataagtctt acaacgtgac caacgtgcgt ttcgctatga agaagtgcat
gtactctatc 240ggcaccttcg tgccgggctc tcagccgggc gaattcaccc
tgggccagat caagtctgaa 300ccgggcaaca cctctaacct ggtgcgtgtg
gtgtctacca actacaacca gcatgctatg 360gtgttcttca aggaagtgta
ccagaaccgt gaaatcttct tcatcaccct gtacggccgt 420accaaggaac
tgacctctga actgaaggaa aacttcatcc gtttctctaa gtctctgggc
480ctgccggaaa accatatcgt gttcccggtg ccgatcgatc agtgcatcga tggc
53414534DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 3 14caggattcta cctctgatct gatcccggct
ccgccgctgt ctaaggtgcc gctgcagcag 60aacttccagg ataaccagtt ccagggcaag
tggtacgtgg tgggcctggc tggcaaccgt 120atcctgcgtc aggatcagca
tccgatgctg atgtacgcta ccatctacga actgaaggaa 180gataagtctt
accaggtgac ctctgtgatc tcttctcata agaagtgcct gtacccgatc
240gctaccttcg tgccgggctc tcagccgggc gaattcaccc tgggcaacat
caagtcttac 300ggcgataagg tgtcttacct ggtgcgtgtg gtgtctacca
actacaacca gcatgctatg 360gtgttcttca agcatgctga taccaactac
gaatctttct ctatcaccct gtacggccgt 420accaaggaac tgacctctga
actgaaggaa aacttcatcc gtttctctaa gtctctgggc 480ctgccggaaa
accatatcgt gttcccggtg ccgatcgatc agtgcatcga tggc
53415207DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 4 15accaccaccc cggctccgcg tccgccgacc
ccggctccga ccatcgcttc tcagccgctg 60tctctgcgtc cggaagcttg ccgtccggct
gctggcggcg ctgtgcatac ccgtggcctg 120gatttcgctt gcgatatcta
catctgggct ccgctggctg gcacctgcgg cgtgctgctg 180ctgtctctgg
tgatcaccct gtactgc 20716339DNAartificialnucleotide sequence
encoding amino acid sequence of SEQ ID NO 5 16ctgcgtgtga agttctctcg
ttctgctgat gctccggctt acaagcaggg ccagaaccag 60ctgtacaacg aactgaacct
gggccgtcgt gaagaatacg atgtgctgga taagcgtcgt 120ggccgtgatc
cggaaatggg cggcaagccg cgtcgtaaga acccgcagga aggcctgtac
180aacgaactgc agaaggataa gatggctgaa gcttactctg aaatcggcat
gaagggcgaa 240cgtcgtcgtg gcaagggcca tgatggcctg taccagggcc
tgtctaccgc taccaaggat 300acctacgatg ctctgcatat gcaggctctg ccgccgcgt
33917132DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 6 17ttctgggtgc gttctaagcg ttctcgtctg
ctgcattctg attacatgaa catgaccccg 60cgtcgtccgg gcccgacccg taagcattac
cagccgtacg ctccgccgcg tgatttcgct 120gcttaccgtt ct
13218123DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 7 18aagcgtggcc gtaagaagct gctgtacatc
ttcaagcagc cgttcatgcg tccggtgcag 60accacccagg aagaagatgg ctgctcttgc
cgtttcccgg aagaagaaga aggcggctgc 120gaa
123191134DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 8 19gcttctgatg aagaaatcca ggatgtgtct
ggcacctggt acctgaaggc tatgaccgtg 60gatacccagg atccgctgtc tctgtacgtg
tctgtgtctc cgatcaccct gaccaccctg 120gaaggcggca acctggaagc
taccgtgacc ctgaaccaga tcggccgttc tcaggaagtg 180ctggctgtgc
tggaaaagac cgatgaaccg ggcaagtaca ccctgtacgg cggcgctcat
240gtggcttaca tccagcgttc tcatgtgaag gatcattaca tcttctactc
tgaaggcgat 300acctggggcg gcccggtgcc gggcgtgtgg ctggtgggcc
gtgatccgaa gaacaacctg 360gaagctctgg aagatttcga aaaggctgct
ggcgctcgtg gcctgtctac cgaatctatc 420ctgatcccgc gtcagtctga
aacctcttct ccgggcacca ccaccccggc tccgcgtccg 480ccgaccccgg
ctccgaccat cgcttctcag ccgctgtctc tgcgtccgga agcttgccgt
540ccggctgctg gcggcgctgt gcatacccgt ggcctggatt tcgcttgcga
tatctacatc 600tgggctccgc tggctggcac ctgcggcgtg ctgctgctgt
ctctggtgat caccctgtac 660tgcttctggg tgcgttctaa gcgttctcgt
ctgctgcatt ctgattacat gaacatgacc 720ccgcgtcgtc cgggcccgac
ccgtaagcat taccagccgt acgctccgcc gcgtgatttc 780gctgcttacc
gttctctgcg tgtgaagttc tctcgttctg ctgatgctcc ggcttacaag
840cagggccaga accagctgta caacgaactg aacctgggcc gtcgtgaaga
atacgatgtg 900ctggataagc gtcgtggccg tgatccggaa atgggcggca
agccgcgtcg taagaacccg 960caggaaggcc tgtacaacga actgcagaag
gataagatgg ctgaagctta ctctgaaatc 1020ggcatgaagg gcgaacgtcg
tcgtggcaag ggccatgatg gcctgtacca gggcctgtct 1080accgctacca
aggataccta cgatgctctg catatgcagg ctctgccgcc gcgt
1134201125DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 9 20gcttctgatg aagaaatcca ggatgtgtct
ggcacctggt acctgaaggc tatgaccgtg 60gatacccagg atccgctgtc tctgtacgtg
tctgtgtctc cgatcaccct gaccaccctg 120gaaggcggca acctggaagc
taccgtgacc ctgaaccaga tcggccgttc tcaggaagtg 180ctggctgtgc
tggaaaagac cgatgaaccg ggcaagtaca ccctgtacgg cggcgctcat
240gtggcttaca tccagcgttc tcatgtgaag gatcattaca tcttctactc
tgaaggcgat 300acctggggcg gcccggtgcc gggcgtgtgg ctggtgggcc
gtgatccgaa gaacaacctg 360gaagctctgg aagatttcga aaaggctgct
ggcgctcgtg gcctgtctac cgaatctatc 420ctgatcccgc gtcagtctga
aacctcttct ccgggcacca ccaccccggc tccgcgtccg 480ccgaccccgg
ctccgaccat cgcttctcag ccgctgtctc tgcgtccgga agcttgccgt
540ccggctgctg gcggcgctgt gcatacccgt ggcctggatt tcgcttgcga
tatctacatc 600tgggctccgc tggctggcac ctgcggcgtg ctgctgctgt
ctctggtgat caccctgtac 660tgcaagcgtg gccgtaagaa gctgctgtac
atcttcaagc agccgttcat gcgtccggtg 720cagaccaccc aggaagaaga
tggctgctct tgccgtttcc cggaagaaga agaaggcggc 780tgcgaactgc
gtgtgaagtt ctctcgttct gctgatgctc cggcttacaa gcagggccag
840aaccagctgt acaacgaact gaacctgggc cgtcgtgaag aatacgatgt
gctggataag 900cgtcgtggcc gtgatccgga aatgggcggc aagccgcgtc
gtaagaaccc gcaggaaggc 960ctgtacaacg aactgcagaa ggataagatg
gctgaagctt actctgaaat cggcatgaag 1020ggcgaacgtc gtcgtggcaa
gggccatgat ggcctgtacc agggcctgtc taccgctacc 1080aaggatacct
acgatgctct gcatatgcag gctctgccgc cgcgt
1125211212DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 10 21caggattcta cctctgatct gatcccggct
ccgccgctgt ctaaggtgcc gctgcagcag 60aacttccagg ataaccagtt ccatggcaag
tggtacgtgg tgggccgtgc tggcaacgtg 120gctctgcgtg aagataagga
tccgccgaag atgcgtgcta ccatctacga actgaaggaa 180gataagtctt
acaacgtgac caacgtgcgt ttcgctatga agaagtgcat gtactctatc
240ggcaccttcg tgccgggctc tcagccgggc gaattcaccc tgggccagat
caagtctgaa 300ccgggcaaca cctctaacct ggtgcgtgtg gtgtctacca
actacaacca gcatgctatg 360gtgttcttca aggaagtgta ccagaaccgt
gaaatcttct tcatcaccct gtacggccgt 420accaaggaac tgacctctga
actgaaggaa aacttcatcc gtttctctaa gtctctgggc 480ctgccggaaa
accatatcgt gttcccggtg ccgatcgatc agtgcatcga tggcaccacc
540accccggctc cgcgtccgcc gaccccggct ccgaccatcg cttctcagcc
gctgtctctg 600cgtccggaag cttgccgtcc ggctgctggc ggcgctgtgc
atacccgtgg cctggatttc 660gcttgcgata tctacatctg ggctccgctg
gctggcacct gcggcgtgct gctgctgtct 720ctggtgatca ccctgtactg
cttctgggtg cgttctaagc gttctcgtct gctgcattct 780gattacatga
acatgacccc gcgtcgtccg ggcccgaccc gtaagcatta ccagccgtac
840gctccgccgc gtgatttcgc tgcttaccgt tctctgcgtg tgaagttctc
tcgttctgct 900gatgctccgg cttacaagca gggccagaac cagctgtaca
acgaactgaa cctgggccgt 960cgtgaagaat acgatgtgct ggataagcgt
cgtggccgtg atccggaaat gggcggcaag 1020ccgcgtcgta agaacccgca
ggaaggcctg tacaacgaac tgcagaagga taagatggct 1080gaagcttact
ctgaaatcgg catgaagggc gaacgtcgtc gtggcaaggg ccatgatggc
1140ctgtaccagg gcctgtctac cgctaccaag gatacctacg atgctctgca
tatgcaggct 1200ctgccgccgc gt 1212221203DNAartificialnucleotide
sequence encoding amino acid sequence of SEQ ID NO 11 22caggattcta
cctctgatct gatcccggct ccgccgctgt ctaaggtgcc gctgcagcag 60aacttccagg
ataaccagtt ccatggcaag tggtacgtgg tgggccgtgc tggcaacgtg
120gctctgcgtg aagataagga tccgccgaag atgcgtgcta ccatctacga
actgaaggaa 180gataagtctt acaacgtgac caacgtgcgt ttcgctatga
agaagtgcat gtactctatc 240ggcaccttcg tgccgggctc tcagccgggc
gaattcaccc tgggccagat caagtctgaa 300ccgggcaaca cctctaacct
ggtgcgtgtg gtgtctacca actacaacca gcatgctatg 360gtgttcttca
aggaagtgta ccagaaccgt gaaatcttct tcatcaccct gtacggccgt
420accaaggaac tgacctctga actgaaggaa aacttcatcc gtttctctaa
gtctctgggc 480ctgccggaaa accatatcgt gttcccggtg ccgatcgatc
agtgcatcga tggcaccacc 540accccggctc cgcgtccgcc gaccccggct
ccgaccatcg cttctcagcc gctgtctctg 600cgtccggaag cttgccgtcc
ggctgctggc ggcgctgtgc atacccgtgg cctggatttc 660gcttgcgata
tctacatctg ggctccgctg gctggcacct gcggcgtgct gctgctgtct
720ctggtgatca ccctgtactg caagcgtggc cgtaagaagc tgctgtacat
cttcaagcag 780ccgttcatgc gtccggtgca gaccacccag gaagaagatg
gctgctcttg ccgtttcccg 840gaagaagaag aaggcggctg cgaactgcgt
gtgaagttct ctcgttctgc tgatgctccg 900gcttacaagc agggccagaa
ccagctgtac aacgaactga acctgggccg tcgtgaagaa 960tacgatgtgc
tggataagcg tcgtggccgt gatccggaaa tgggcggcaa gccgcgtcgt
1020aagaacccgc aggaaggcct gtacaacgaa ctgcagaagg ataagatggc
tgaagcttac 1080tctgaaatcg gcatgaaggg cgaacgtcgt cgtggcaagg
gccatgatgg cctgtaccag 1140ggcctgtcta ccgctaccaa ggatacctac
gatgctctgc atatgcaggc tctgccgccg 1200cgt
120323209PRTartificialsecreted anti-CTLA-4 antagonist 23Met Asp Phe
Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile
Met Ser Arg Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro 20 25 30Pro
Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe 35 40
45Gln Gly Lys Trp Tyr Val Val Gly Leu Ala Gly Asn Arg Ile Leu Arg
50 55 60Gln Asp Gln His Pro Met Leu Met Tyr Ala Thr Ile Tyr Glu Leu
Lys65 70 75 80Glu Asp Lys Ser Tyr Gln Val Thr Ser Val Ile Ser Ser
His Lys Lys 85 90 95Cys Leu Tyr Pro Ile Ala Thr Phe Val Pro Gly Ser
Gln Pro Gly Glu 100 105 110Phe Thr Leu Gly Asn Ile Lys Ser Tyr Gly
Asp Lys Val Ser Tyr Leu 115 120 125Val Arg Val Val Ser Thr Asn Tyr
Asn Gln His Ala Met Val Phe Phe 130 135 140Lys His Ala Asp Thr Asn
Tyr Glu Ser Phe Ser Ile Thr Leu Tyr Gly145 150 155 160Arg Thr Lys
Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe 165 170 175Ser
Lys Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro 180 185
190Ile Asp Gln Cys Ile Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu
195 200 205Lys24630DNAartificialnucleotide sequence encoding the
amino acid sequence of SEQ ID NO 23 24atggattttc aggtgcagat
tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agacaggact ccacctctga
tctcatcccc gcacctccgc ttagcaaggt tcccttgcaa 120cagaattttc
aggacaatca attccagggg aaatggtacg tagtggggct cgccggcaac
180agaatcctga gacaagacca gcacccaatg ctgatgtacg ctacgatcta
cgagttgaag 240gaagacaagt catatcaagt aacaagcgtt atttcttctc
acaaaaaatg cctgtatcca 300atcgctacat ttgtccctgg ttcccagccc
ggggaattta cccttggcaa catcaagtct 360tatggtgata aagtgtccta
tctggtgaga gttgtctcta ccaattacaa tcagcacgct 420atggtcttct
tcaaacatgc cgatacaaat tacgaaagct tcagtatcac tctgtatgga
480aggactaaag aattgactag cgagcttaaa gagaacttca tacggttcag
caaaagcctg 540gggctccccg agaaccatat tgtgtttccc gtacctatag
atcagtgcat tgacggcagt 600gcatggtctc acccccagtt cgagaaatga
63025445PRTartificialsecreted anti-CTLA-4 antagonist 25Met Asp Phe
Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile
Met Ser Arg Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro 20 25 30Pro
Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe 35 40
45Gln Gly Lys Trp Tyr Val Val Gly Leu Ala Gly Asn Arg Ile Leu Arg
50 55 60Gln Asp Gln His Pro Met Leu Met Tyr Ala Thr Ile Tyr Glu Leu
Lys65 70 75 80Glu Asp Lys Ser Tyr Gln Val Thr Ser Val Ile Ser Ser
His Lys Lys 85 90 95Cys Leu Tyr Pro Ile Ala Thr Phe Val Pro Gly Ser
Gln Pro Gly Glu 100 105 110Phe Thr Leu Gly Asn Ile Lys Ser Tyr Gly
Asp Lys Val Ser Tyr Leu 115 120 125Val Arg Val Val Ser Thr Asn Tyr
Asn Gln His Ala Met Val Phe Phe 130 135 140Lys His Ala Asp Thr Asn
Tyr Glu Ser Phe Ser Ile Thr Leu Tyr Gly145 150 155 160Arg Thr Lys
Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe 165 170 175Ser
Lys Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro 180 185
190Ile Asp Gln Cys Ile Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu
195 200 205Lys Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr
Cys Pro 210 215 220Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310
315 320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425
430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys 435 440
445261338DNAartificialnucleotide sequence encoding amino acid
sequence of SEQ ID NO 25 26atggattttc aggtgcaaat cttttctttc
cttctgatta gcgcttctgt gatcatgtca 60aggcaagatt caacttccga tctgatcccg
gcgccaccac tctccaaggt accactgcag 120cagaactttc aggataacca
gtttcagggg aaatggtacg tagtgggcct tgccggaaac 180cgaatcctgc
ggcaggatca acaccccatg ctgatgtacg ctaccatcta tgagctgaag
240gaagataaga gttatcaggt gacgagcgtc atctcttccc acaaaaagtg
cctgtatccc 300attgccacat tcgtgccggg cagtcaaccg ggcgagttca
ccctcgggaa catcaaatcc 360tacggcgata aggtaagtta cttggtcagg
gtcgtgtcta caaactacaa ccaacacgcc 420atggtttttt tcaagcacgc
tgatacaaac tacgagagct tctcaatcac cttgtacggt 480cggaccaaag
aactgacgtc tgagcttaag gagaatttta tacgcttctc aaagagcctg
540gggctgccag aaaaccacat cgttttccca gtgccaatcg accagtgtat
cgatggctct 600gcctggtccc acccacagtt tgagaaagat ccagctgaac
caaagagtcc agacaagact 660cacacatgtc caccttgccc cgccccagaa
ttgttgggag gcccctctgt gtttctcttc 720cctcctaagc caaaagacac
tctgatgata tcacggaccc cagaggttac ttgcgttgtc 780gtcgatgtga
gtcacgaaga tcctgaggtc aagtttaact ggtacgtaga tggggttgag
840gtgcataacg caaagaccaa acctcgcgag gaacagtata acagtacata
tcgcgtggta 900tccgtgctca ccgtcctcca tcaagactgg ttgaatggaa
aggaatacaa gtgcaaagtt 960tctaacaaag ccctgccagc gcccatcgaa
aagactatct ctaaagccaa gggccaacct 1020cgcgaacctc aagtgtacac
ccttcctccc agccgggatg aactgaccaa aaaccaagtg 1080agcctgacat
gtctggtgaa gggtttctat ccctctgata ttgcggttga atgggaaagc
1140aacggacagc ccgaaaacaa ctacaagact acaccccccg ttttggattc
cgatgggagt 1200tttttcttgt attctaagct gaccgttgat aaaagtaggt
ggcagcaggg caatgtgttt 1260tcatgtagcg tgatgcacga ggcccttcac
aaccactata cccagaagag tcttagcctc 1320agccctggaa agaagtga
13382710PRTartificialStrep-tag II 27Ser Ala Trp Ser His Pro Gln Phe
Glu Lys1 5 1028188PRTartificialsecreted anti-CTLA-4 antagonist
without leader sequence 28Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala
Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe Gln Asp Asn
Gln Phe Gln Gly Lys Trp Tyr 20 25 30Val Val Gly Leu Ala Gly Asn Arg
Ile Leu Arg Gln Asp Gln His Pro 35 40 45Met Leu Met Tyr Ala Thr Ile
Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Gln Val Thr Ser Val Ile
Ser Ser His Lys Lys Cys Leu Tyr Pro Ile65 70 75 80Ala Thr Phe Val
Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95Ile Lys Ser
Tyr Gly Asp Lys Val Ser Tyr Leu Val Arg Val Val Ser 100 105 110Thr
Asn Tyr Asn Gln His Ala Met Val
Phe Phe Lys His Ala Asp Thr 115 120 125Asn Tyr Glu Ser Phe Ser Ile
Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys
Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro
Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 180
18529424PRTartificialsecreted anti-CTLA-4 antagonist without leader
sequence 29Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser
Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly
Lys Trp Tyr 20 25 30Val Val Gly Leu Ala Gly Asn Arg Ile Leu Arg Gln
Asp Gln His Pro 35 40 45Met Leu Met Tyr Ala Thr Ile Tyr Glu Leu Lys
Glu Asp Lys Ser Tyr 50 55 60Gln Val Thr Ser Val Ile Ser Ser His Lys
Lys Cys Leu Tyr Pro Ile65 70 75 80Ala Thr Phe Val Pro Gly Ser Gln
Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95Ile Lys Ser Tyr Gly Asp Lys
Val Ser Tyr Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln
His Ala Met Val Phe Phe Lys His Ala Asp Thr 115 120 125Asn Tyr Glu
Ser Phe Ser Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr
Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150
155 160Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys
Ile 165 170 175Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Asp
Pro Ala Glu 180 185 190Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro 195 200 205Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 210 215 220Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val225 230 235 240Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 245 250 255Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 260 265
270Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
275 280 285Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu 290 295 300Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg305 310 315 320Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys 325 330 335Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 340 345 350Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 355 360 365Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 370 375 380Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser385 390
395 400Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 405 410 415Leu Ser Leu Ser Pro Gly Lys Lys 42030178PRThuman
30Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1
5 10 15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp
Tyr 20 25 30Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys
Asp Pro 35 40 45Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp
Lys Ser Tyr 50 55 60Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys
Asp Tyr Trp Ile65 70 75 80Arg Thr Phe Val Pro Gly Cys Gln Pro Gly
Glu Phe Thr Leu Gly Asn 85 90 95Ile Lys Ser Tyr Pro Gly Leu Thr Ser
Tyr Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala
Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125Asn Arg Glu Tyr Phe
Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu
Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155
160Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175Asp Gly
* * * * *