U.S. patent application number 15/818225 was filed with the patent office on 2018-03-15 for polypeptide that binds aberrant cells and induces apoptosis.
This patent application is currently assigned to APO-T B.V.. The applicant listed for this patent is APO-T B.V.. Invention is credited to Johan Renes, Ralph Alexander Willemsen.
Application Number | 20180071398 15/818225 |
Document ID | / |
Family ID | 45771866 |
Filed Date | 2018-03-15 |
United States Patent
Application |
20180071398 |
Kind Code |
A1 |
Willemsen; Ralph Alexander ;
et al. |
March 15, 2018 |
POLYPEPTIDE THAT BINDS ABERRANT CELLS AND INDUCES APOPTOSIS
Abstract
Described are proteinaceous molecules comprising at least a
domain that comprises an amino acid sequence that specifically
binds to an MHC-peptide complex on an aberrant cell, functionally
connected with a substance that induces apoptosis in aberrant
cells, but not in normal cells. These proteinaceous molecules are
preferably used in selectively modulating biological processes. The
provided proteinaceous molecules are of particular use in
pharmaceutical compositions for the treatment of diseases related
to cellular aberrancies, such as cancers.
Inventors: |
Willemsen; Ralph Alexander;
(Rotterdam, NL) ; Renes; Johan; (Amersfoort,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APO-T B.V. |
Oss |
|
NL |
|
|
Assignee: |
APO-T B.V.
Oss
NL
|
Family ID: |
45771866 |
Appl. No.: |
15/818225 |
Filed: |
November 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13976974 |
Nov 14, 2013 |
9821073 |
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PCT/NL11/50891 |
Dec 22, 2011 |
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15818225 |
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61460212 |
Dec 27, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
A61K 47/6851 20170801; C07K 2317/32 20130101; C07K 16/2833
20130101; C07K 2317/55 20130101; C07K 2317/56 20130101; A61P 35/00
20180101; A61K 47/64 20170801; C07K 2317/21 20130101; C07K 2317/35
20130101; C07K 2317/34 20130101; C07K 16/30 20130101; C07K 2317/73
20130101; A61K 45/06 20130101; C07K 2319/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; A61K 45/06 20060101
A61K045/06 |
Claims
1.-21. (canceled)
22. A molecule comprising: at least one domain that comprises an
amino acid sequence that specifically binds to an MHC-peptide
complex, wherein the peptide within the MHC-peptide complex
comprises a MAGE peptide, said at least one domain functionally
connected with a substance that induces apoptosis in aberrant
cells, but not normal cells.
23. The molecule of claim 22, wherein the substance is an
apoptosis-inducing polypeptide or protein and the at least one
domain are linked via peptide bonds.
24. The molecule of claim 23, which comprises a single polypeptide
chain.
25. The molecule of claim 22, wherein the at least one domain
specifically binds an MHC-1-peptide complex.
26. The molecule of claim 22, wherein the substance is apoptin or a
fragment and/or derivative thereof able to induce apoptosis in an
aberrant cells, but not in a normal cell.
27. The molecule of claim 22, wherein the substance comprises a
statin.
28. The molecule of claim 22, wherein the at least one domain is
linked to the substance through a non-peptide bond.
29. A polynucleotide encoding a domain that comprises an amino acid
sequence that specifically binds to an MHC-peptide complex.
30. A polynucleotide encoding the molecule of claim 23.
31. A vector comprising the polynucleotide of claim 30 together
with elements for expression in a suitable host cell.
32. A host cell comprising the polynucleotide of claim 29
integrated into the host cell.
33. A method for producing a molecule, the method comprising:
culturing the host cell of claim 32, expressing the polynucleotide,
and harvesting the resulting molecule.
34. A pharmaceutical composition comprising: the polypeptide of
claim 22, and a suitable diluent and/or excipient.
35. The pharmaceutical composition of claim 34, further comprising:
a conventional cytostatic and/or tumoricidal agent.
36. A method of treating a subject for cancer, the method
comprising: utilizing the molecule of claim 22 in the treatment of
the subject's cancer.
37. The method according to claim 36, wherein the molecule is
utilized as an adjuvant treatment of the cancer.
38. The method according to claim 36, wherein the molecule is
utilized in a combination chemotherapy treatment of cancer.
39. A method of treating a subject for cancer, the method
comprising: administering the pharmaceutical composition of claim
34 to the subject so as to treat the cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 13/976,974, filed Nov. 14, 2013, U.S. Pat. No.
9,821,073 (Nov. 21, 2017), and which is a national phase entry
under 35 U.S.C. .sctn. 371 of International Patent Application
PCT/NL2011/050891, filed Dec. 22, 2011, designating the United
States of America and published in English as International Patent
Publication WO 2012/091563 A1 on Jul. 5, 2012, which claims the
benefit under Article 8 of the Patent Cooperation Treaty and under
35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent Application Ser.
No. 61/460,212, filed Dec. 27, 2010, the contents of the entirety
of each of which is incorporated herein by this reference.
TECHNICAL FIELD
[0002] The application relates to the field of biotherapeutics. It
also relates to the field of tumor biology. More in particular, it
relates to specific binding molecules that induce cell death, in
particular, programmed cell death (apoptosis) in aberrant cells
such as tumor cells. More specifically, one or multiple antibody
variable fragments connected with a cell death-inducing agent such
as apoptin are provided that specifically target MHC-peptide
complexes on aberrant cells, thereby delivering a cell
death-inducing agent such as apoptin that induces apoptosis upon
uptake of the specific binding molecule. It also relates to the use
of these apoptosis-inducing binding molecules in selectively
killing cancer cells and other aberrant cells.
BACKGROUND
[0003] Since the sixties of the last century, it has been proposed
to use the specific binding power of the immune system (T cells and
antibodies) to selectively kill tumor cells but leave alone the
normal cells in a patient's body. Many tumor antigens that could be
targeted by, in particular, antibodies, like carcino-embryonic
antigen (CEA), alpha-fetoprotein (AFP) and so on, have been
suggested since those days, but for essentially all of these
antigens, expression is associated with normal tissue as well.
Thus, so far, selective killing of aberrant cells has been an
elusive goal.
[0004] The primary immunological function of MHC molecules is to
bind and to "present" antigenic peptides to form an MHC-peptide
(MHC-p) complex on the surface of cells for recognition and binding
by antigen-specific T-cell receptors (TCRs) of lymphocytes.
Antigenic peptides are also referred to as epitopes, both of which
have basically the same meaning throughout the application. Two
classes of MHC-p complexes can be distinguished with regard to
their function:
[0005] (i) MHC class I-p complexes can be expressed by almost all
nucleated cells in order to attract CD8.sup.- cytotoxic T cells,
and
[0006] (ii) MHC class II-p complexes are constitutively expressed
only on so-called antigen-presenting cells (APCs), such as B
lymphocytes, macrophages or dendritic cells (DCs).
[0007] MHC class I-p complexes are composed of a variable heavy
chain, an invariable .beta.-microglobulin and an antigenic peptide.
The MHC class II molecules are characterized by distinctive .alpha.
and .beta. polypeptide subunits that combine to form .alpha..beta.
heterodimers characteristic of mature MHC class II molecules.
Differential structural properties of MHC class I and class II
molecules account for their respective roles in activating
different populations of T lymphocytes. Cytotoxic T.sub.C
lymphocytes (CTLs) bind antigenic peptides presented by MHC class I
molecules. Helper T.sub.H lymphocytes bind antigenic peptides
presented by MHC class II molecules. MHC class I and class II
molecules differentially bind CD8 and CD4 cell adhesion molecules.
MHC class I molecules are specifically bound by CD8 molecules
expressed on CTLs, whereas MHC class II molecules are specifically
bound by CD4 molecules expressed on helper T.sub.H lymphocytes.
[0008] The sizes of the antigenic peptide-binding pockets of MHC
class I and class II molecules differ; class I molecules bind
smaller antigenic peptides, typically eight to ten amino acid
residues in length, whereas class II molecules bind larger
antigenic peptides, typically 13 to 18 amino acid residues in
length.
[0009] In humans, MHC molecules are termed human leukocyte antigens
(HLA). HLA-associated peptides are short, encompassing typically 9
to 25 amino acid residues. Humans synthesize three different types
of class I molecules designated HLA-A, HLA-B, and HLA-C. Human
class II molecules are designated HLA-D, e.g., HLA-DR.
[0010] The MHC expressed on all nucleated cells of humans and of
animals plays a crucial role in immunological defense against
pathogens and cancer. The transformation of normal cells to
aberrant cancer cells involves several major changes in gene
expression. This results in profound changes in the antigenic
composition of cells. It is well established that new antigenic
entities are presented as MHC-restricted tumor antigens. As such,
the MHC class I and MHC class II systems may be seen as nature's
proteomic scanning chips, continuously processing intracellular
proteins, generating antigenic peptides for presentation on the
cell surface. If these antigenic peptides elicit an immune
reactivity, the transformed cells are killed by the cellular immune
system. However, if the transformed cells resist immune-mediated
cell killing, cancer may develop.
[0011] Antibodies that bind MHC class I molecules on various cell
types have been studied in detail for their mode of action. Mouse
monoclonal antibodies that bind the MHC class I .alpha.1 domain of
the MHC class I .alpha. chain induce apoptosis in activated T
cells, but not in resting T cells. Other reports mention antibodies
specific for, e.g., the .alpha.3 domain of MHC class I, which
induce growth inhibition and apoptosis in B-cell-derived cancer
cells. However, in this case, a secondary cross-linking antibody
was required for the induction of apoptosis (A. E. Pedersen et al.,
Exp. Cell Res. 1999, 251:128-34).
[0012] Antibodies binding to .beta.2-microglobulin (.beta.2-M), an
essential component of the MHC class I molecules, also induce
apoptosis. Several hematologic cancer cells treated with
anti-.beta.2M antibodies were killed efficiently, both in vitro and
in vivo (Y. Cao et al., Br. J. Haematol. 2011, 154:111-121).
[0013] Thus, it is known that binding of MHC class I or MHC class
II molecules by several anti-MHC antibodies can have an
apoptosis-inducing effect. However, the therapeutic application of
these anti-MHC antibodies has been hampered by the lack of target
cell specificity. Since these antibodies are directed primarily
against a constant domain of the MHC molecule, the cell surface
expression of the MHC constant domain determines whether or not a
cell can be triggered by the antibody to undergo apoptosis. Because
MHC class I and MHC class II molecules are expressed on both normal
and aberrant cells, it is clear that these antibodies cannot
discriminate between normal and aberrant cells. As a consequence,
their therapeutic value is significantly reduced, if not abolished
by the side effects caused by unwanted apoptosis of healthy cells.
According to the invention, antibodies that specifically recognize
MHC-presented antigenic peptides derived from cancer antigens
would, therefore, dramatically expand the therapeutic repertoire,
if they could be shown to have anti-cancer cell activity. In
addition, current methods to induce apoptosis via MHC class I or
MHC class II may depend on external cross-linking of anti-MHC
antibodies.
[0014] Obtaining antibodies binding to MHC-p complexes and not
binding to MHC molecules not loaded with the antigenic peptide
remains a laborious task and several failures have been reported.
The first available antibodies have been obtained after
immunization of mice with recombinant MHC-p complexes or
peptide-loaded TAP-deficient antigen-presenting cells. More
recently, antibodies have been obtained by selection from
phage-antibody libraries made from immunized transgenic mice or by
selection from completely human antibody phage libraries.
Immunization with MHC-p complexes is extremely time consuming.
Moreover, antibodies of murine origin cannot be used repetitively
in patients because of the likely development of a human anti-mouse
antibody response (so-called anti-drug antibodies, ADA). Antibodies
derived from phage display, in general, display low affinity for
the antigen and thus may require additional modifications before
they can be used efficiently. According to the invention, the
antibody specificities are preferably selected through phage (or
yeast) display, whereby an MHC molecule loaded with a
cancer-related peptide is presented to the library. Details are
given in the experimental part. The antibody specificities
according to the invention are checked for specificity to the
MHC-peptide complex and should not recognize (to any significant
extent) MHC loaded with irrelevant peptides or the peptides by
themselves.
[0015] Cancer is caused by oncogenic transformation in aberrant
cells, which drives uncontrolled cell proliferation, leading to
misalignment of cell-cycle checkpoints, DNA damage and metabolic
stress. These aberrations should direct tumor cells toward an
apoptotic path that has evolved in multi-cellular animals as a
means of eliminating abnormal cells that pose a threat to the
organism. Indeed, most transformed cells or tumorigenic cells are
killed by apoptosis. However, occasionally, a cell with additional
mutations that enable avoidance of apoptotic death survives, thus
enabling its malignant progression. Thus, cancer cells can grow,
not only due to imbalances in proliferation and/or cell cycle
regulation, but also due to imbalances in their apoptosis
machinery. Imbalances like, for example, genomic mutations
resulting in non-functional apoptosis-inducing proteins or
over-expression of apoptosis-inhibiting proteins, form the basis of
tumor formation. Fortunately, even cells that manage to escape the
apoptosis signals this way when activated by their aberrant
phenotype, are still primed for eradication from the organism.
Apoptosis in these aberrant cells can still be triggered upon
silencing or overcoming the apoptosis-inhibiting signals induced by
mutations. Traditional cancer therapies can activate apoptosis, but
they do so indirectly and often encounter tumor resistance. Direct
and selective targeting of key components of the apoptosis
machinery in these aberrant cells is a promising strategy for
development of new anti-tumor therapeutics. Selective activation of
the apoptosis pathway would allow for halting tumor growth and
would allow for induction of tumor regression.
[0016] A disadvantage of many, if not all, anti-tumor drugs
currently on the market or in development, which are based on
targeting the apoptosis machinery, is that these drugs do not
discriminate between aberrant cells and healthy cells. This
non-specificity bears a challenging risk for drug-induced adverse
events. Examples of such unwanted side effects are well known to
the field: radiotherapy and chemotherapeutics induce apoptosis only
as a secondary effect of the damage they cause to vital cellular
components. Not only aberrant cells are targeted, though, in fact,
most proliferating cells including healthy cells respond to the
apoptosis-stimulating therapy. Therefore, a disadvantage of current
apoptosis-inducing compounds is their non-selective nature, which
reduces their potential.
[0017] In an earlier application (WO2007/073147; Apoptosis-inducing
protein complexes and therapeutic use thereof; incorporated herein
by reference), it is disclosed that a polypeptide complex achieves
the goal of (specifically) killing, e.g., tumor cells by
specifically targeting these cells and, as a result, induces
apoptosis in these tumor cells. Although it is undesirable to be
bound by theory, at present, it is believed that this is the result
of cross-linking of cell-surface-expressed protein-protein
complexes by multiple interactions with the multivalent polypeptide
complex of that invention.
[0018] Two interlinked signaling pathways control apoptosis
activation. Intracellular signals, such as DNA damage, drive
apoptosis primarily through the intrinsic pathway, controlled by
the Bcl-2 protein family. Extracellular signals, usually generated
by cytotoxic cells of the immune system such as natural killer
cells or cytotoxic T cells, trigger apoptosis mainly through the
extrinsic pathway. Both pathways stimulate caspases with
apoptosis-inducing activity. Caspases are a family of cysteine
proteases, which are present in most cells as pro-caspases and
which are activated through the so-called caspase cascade.
Apoptotic signals first stimulate upstream initiator caspases
(amongst others, caspases 8, 9 and 10) by recruiting them into
specific signaling complexes that promote their multimerization. In
turn, these caspases in signaling complexes activate downstream
effector caspases (including caspases 3, 6 and 7) by proteolytic
processing. These effector caspases then, in turn, process various
cellular proteins, resulting in the apoptotic cell death
program.
[0019] Some viruses (or at least some of their proteins), such as
chicken anemia virus (CAV), parvovirus minute virus of mice (MVM),
engineered herpes simplex virus, reovirus, vesicular stomatitis
virus, adenovirus type 2 and poxvirus such as vaccinia, can
selectively and preferentially kill tumor cells. These viruses do
so through activation of the apoptosis machinery of the aberrant
cell infected by the virus. The viruses are able to specifically
provide the effective apoptosis-inducing death signal, which can
interact with one or more of the derailed cancer processes.
Fortunately, these viruses (or their proteins) have the ability to
efficiently target cell death program in aberrant cells, although
this cell death program might be derailed as a consequence of its
aberrant nature. Two oncolytic virus-based therapies are tested in
clinical trials: Reolysin, which is a reovirus, and Onyx-015, which
is an adenovirus deletion mutant. The various clinical trials
revealed that the therapeutic agents were selective for cancer
cells, but therapeutic potency was limited. In general, anti-tumor
gene therapy has largely failed to date in patients owing to
inefficient delivery of the gene to sufficient numbers of cancer
cells locally and systemically. Development of new generation
anti-tumor drugs should, therefore, focus on improved anticancer
potency, improved efficacy of delivery and improved systemic
spread.
[0020] Interestingly, proteins derived from several of these
viruses, i.e., CAV-derived apoptosis-inducing apoptin, adenovirus
early region 4 open reading frame (E4orf4) and
parvovirus-H1-derived non-structural protein 1 (NS1), were
identified as agents that are able to induce aberrant-cell
apoptosis. For example, apoptin was shown to be the main aberrant
cell-specific apoptosis-inducing factor of CAV. In addition to
these apoptosis-inducing proteins identified in these viruses, new
apoptosis-inducing proteins were identified that are not part of
viruses' genomes but that are also able to induce cell death
specifically in aberrant cells. Examples are human
.alpha.-lactalbumin made lethal to tumor cells (HAMLET), human
cytokines melanoma differentiation-associated gene-7 (mda-7) and
tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL).
[0021] The ability of these viral proteins apoptin, E4orf4 and NS1
and these non-viral cellular proteins HAMLET, TRAIL and mda-7 to
induce apoptosis in aberrant cells renders them with a high potency
for beneficial incorporation in anti-tumor therapies.
[0022] Parvovirus-H1 NS1 protein induces cell death in glioma
cells. The tumor-selective apoptosis-inducing activity of NS1 is
related to its interaction with the catalytic subunit of casein
kinase II (CKII.alpha.). Formation of NS1-CKII.alpha. complexes
points to interference by NS1 with intracellular signaling
processes (Noteborn, Eur. J. Pharm., 2009). As a result of the
formed NS1-CKII.alpha. complexes, CKII.alpha.-dependent
cytoskeletal changes occur followed by apoptosis. Parvovirus-H1
infections induce characteristic changes within the cytoskeleton
filaments of tumor cells, which results finally in the degradation
of actin fibers and the appearance of so-called actin patches.
[0023] Loss of p53 functioning is related to tumor formation and is
at the basis of resistance of tumors to various anticancer
therapies. The adenovirus-derived protein E4orf4 selectively kills
tumor cells independent of p53 (Noteborn, Eur. J. Pharm., 2009).
Like parvovirus-H1-derived protein NS1, E4orf4 expression results
in deregulation of the cytoskeleton. E4orf4-induced cell death is
not dependent on classical caspase pathways, and E4orf4 circumvents
Bcl-2 blockage of apoptosis and does not require release of
mitochondrial cytochrome c. Seemingly, E4orf4 is able to trigger
apoptosis in aberrant cells via an alternative cell death process
not present in non-aberrant cells.
[0024] Human .alpha.-lactalbumin made lethal to tumor cells
(HAMLET) is a structural derivative of .alpha.-lactalbumin, a main
protein of human milk. HAMLET can induce apoptosis in a
tumor-selective manner (Noteborn, Eur. J. Pharm., 2009). The
precursor of HAMLET is .alpha.-lactalbumin, which undergoes
structural changes upon binding of oleic acid and subsequent
release of calcium ions. HAMLET can specifically kill aberrant
cells of skin papillomas, glioblastoma tumors, and bladder cancers
by efficient uptake, leaving healthy tissue unaltered. HAMLET acts
on the caspase pathways due to stimulated release of cytochrome c
from the mitochondria. In the nuclei of tumor cells, HAMLET
associates with histones resulting in an irreversible disruption of
the chromatin organization. This seems the key event responsible
for the tumor-cell killing activity of HAMLET, apart from its
ability to activate 20S proteasomes. HAMLET induces tumor-selective
apoptosis in a p53-independent manner.
[0025] Melanoma differentiation-associated gene-7 (mda-7;
interleukin 24), an interleukin-10 family member, induces apoptosis
in various cancer cells dependent on caspases (Noteborn, Eur. J.
Pharm., 2009). For example, apoptosis-inducing activity of mda-7
upon down-regulation of survival signals such as Bcl-2 and Akt by
mda-7 is seen in breast cancer cells when adenoviral-induced mda-7
is used. Also secreted mda-7 exposes anti-tumor cell activity on
distant tumor cells. Specificity of mda-7 apoptosis-inducing
activity is based on the activation of the FasL/TRAIL pathways.
Mda-7 has been proven effective pre-clinically in treatment of
subcutaneous ovarian cancer xenografts and lung tumor xenografts
(combination therapy), when adenovirus-expressing mda-7 was used. A
clinical phase I trial revealed that subsets of tumor cells are
resistant to mda-7, leaving substantial room for further
improvement of therapies based on proteins bearing
apoptosis-inducing activity.
[0026] The tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) induces both p53-dependent and p53-independent apoptosis in
tumor cells (Noteborn, Eur. J. Pharm., 2009). TRAIL activates the
extrinsic apoptosis pathway leading to caspase 8 and subsequently
amongst other caspase-3 activation. Subsequently, TRAIL-induced
apoptosis activates the intrinsic apoptosis pathway. One of the
first steps in TRAIL-induced apoptosis is the binding of TRAIL to
death receptors DR4 and DR5. TRAIL's apoptosis activity is
selective for tumor cells but the diversity of tumor cells
susceptible to TRAIL-induced apoptosis is limited. This is perhaps
due to the fact that TRAIL signaling also activates NF-.kappa.B,
which induces anti-apoptotic regulators. In addition or
alternatively, TRAIL resistance of several types of tumor cells may
be due to the fact that these tumor cells over-express
anti-apoptosis protein FLIP or Bcl-2.
[0027] The CAV-derived apoptin is a viral protein with
apoptosis-inducing activity toward a broad range of human aberrant
cell types but not toward normal, non-transformed human diploid
cells including primary human hepatocytes and stem cells. A broad
variety of tumor cell types is susceptible to apoptin's
apoptosis-inducing activity. This apoptin activity can be triggered
by induced transformation of cells. These two observations point to
regulation of the apoptosis pathway by apoptin during an early
stage of the cell transformation process. The specificity of
apoptin for tumor cells may be related to its multimeric nature
when in its active form, its interaction with chromatin structures
in tumor cells, its selective phosphorylation in malignant cells,
and its ability to elevate ceramide levels in tumor cells, which is
a tumor suppressor activity. This latter activity is indicative for
an important role of sphingolipids in apoptin-induced apoptosis.
Apoptin induces apoptosis also by acting on and interfering with
the cell cycle processes. That is to say, apoptin acts mainly via
interaction with the anaphase-promoting complex/cyclosome complex,
inducing G2/M cell cycle arrest resulting in p73/PUMA-mediated
apoptosis. Cytochrome c release and activation of the central
caspase pathways are involved in apoptin-induced cell death. The
selectivity of apoptin's apoptosis-inducing activity for tumor
cells is p53 independent and, in several tumor cell types, is not
sensitive to Bcl-xl and even stimulated by Bcl-2. In normal cells,
apoptin is found located mainly in the cytoplasm. In transformed
cells and in malignant cells characterized by metaplasia,
hyperplasia or dysplasia, apoptin localizes (also) in the nucleus
(Danen-van Oorschot et al., 1997).
[0028] Application of apoptin biology has been tested for its
efficiency in selectively killing tumor cells in a series of in
vitro and in vivo cancer models. Thus far, apoptin has shown a
beneficial apoptosis-inducing effect pre-clinically in the context
of hepato-carcinoma, breast carcinoma, lung cancer, liver cancer
and prostate cancer. Exposing tumor cells to apoptin resulted in a
slowdown of tumor growth or even a complete regression of tumors,
when delivered to cancer cells intra-tumoral via a non-replicative
adenovirus, for the treatment of hepatoma (when part of the
Fowl-pox virus genome) (Li et al., Int. J. Cancer, 2006).
Beneficial effects of apoptin treatment were also reported for
Lewis lung carcinoma, when delivered to the aberrant cells as part
of plasmid DNA and for hepato-carcinoma, when applying the Asor-DNA
delivery approach. For lung tumors, cervix carcinomas, gastric
cancer and hepato-carcinomas, apoptin proved effective when
recombinant apoptin was used complexed with a polypeptide for
tunneling apoptin into targeted cells, i.e., the protein transfer
domain TAT protein of HIV or PTD4. Apoptin was beneficial in the
treatment of osteosarcoma and prostate cancer, when combined in
combinatorial therapeutic approaches (Olijslagers et al., Basic
Clin. Pharmacol. Toxicol., 2007). On the other side, apoptin has
been proven to be inactive regarding its apoptosis-inducing
activity in normal lymphoid cells, dermal cells, epidermal cells,
endothelial cells and smooth muscle cells, providing further
insight in the cancer cell specificity of apoptin (Danen-van
Oorschot et al. 1997).
[0029] Apoptin, comprising 121 amino-acid residues, consists of
proline-rich regions, two basic C-terminal clusters K82-R89 and
R111-R120 and, over all, contains a high percentage of serine and
threonine residues. The two basic clusters comprise the apoptin
nuclear localization signal in the apoptin 81-121 amino-acid
residues fragment. These clusters form a tumor-selective apoptosis
domain, regulated by phosphorylation of threonine residue 108
(additionally, apoptin comprises four serine phosphorylation sites
in total). A second tumor-selective apoptosis domain is located at
the N-terminus of apoptin and is a hydrophobic domain, involved in
apoptin multimerization (apoptin amino-acid residues 1-69) and
comprising interaction sites for other, possibly numerous proteins.
Multimerization of apoptin results in protein globules,
predominantly spherical in shape, consisting of approximately 30
apoptin molecules each. These homogenous oligomerized apoptin
globules have tumor-selective apoptosis-inducing activity. The
apoptin is approximately 30 mers and can be soluble in nature, or
can be insoluble.
[0030] Based on the secondary structure prediction results of five
different algorithms, feeding the algorithms with the full-length
apoptin sequence 1-121 (SEQ ID NO:3), the apoptin amino-acid
sequence .sup.32Glu-Leu.sup.46 (e.g., amino acids 32-46 of SEQ ID
NO:3) encompasses two predicted beta-strands:
.sup.32Glu-Ile-Arg-Ile.sup.35 (amino acids 32-35 of SEQ ID NO:3)
and .sup.40Ile-Thr-Ile-Thr-Leu-Ser.sup.45 (amino acids 40-45 of SEQ
ID NO:3), of which the latter is possibly extended with .sup.39Gly
and/or with Leu.sup.46. Circular dichroism spectropolarimetry
experiments with an apoptin-His6 construct indeed revealed that
apoptin multimers built up of approximately 30 mers have adopted
beta-sheet secondary structure to a small extent. The consensus
beta-strands allow for formation of an anti-parallel
intra-molecular beta-sheet in apoptin molecules. This beta-sheet
encompasses two beta-strands: strand a, residues
.sup.32-Glu-Ile-Arg-Ile-.sup.35 (amino acids 32-35 of SEQ ID NO:3),
and strand b, residues .sup.40-Ile-Thr-Ile-Thr-.sup.43 (amino acids
40-43 of SEQ ID NO:3), linked by residues
.sup.36-Gly-Ile-Ala-Gly-.sup.39 (amino acids 36-39 of SEQ ID NO:3).
Amino-acid residues Ile33, Ile35, Ile40 and Ile42 form a
hydrophobic face at one side of the intra-molecular beta-sheet;
Glu32, Arg34, Thr41 and Thr43 form a charged and hydrophilic
opposite face of the same beta-sheet. Thus, hydrophobic side chains
of all Ile residues are located at one side of the beta-sheet, with
all hydrophilic and charged side chains pointing outward at the
opposite side of the anti-parallel beta-sheet. With eight amino
acid residues in beta-sheet conformation in apoptin 30-mer
globules, in theory, 6.6% beta-sheet content could be determined
with a CD measurement. With a hydrophobic face and a
charged/hydrophilic face, protein surfaces are formed at apoptin
that are accessible for incorporation in an inter-molecular
amyloid-like structure build up by, apparently, approximately 30
apoptin molecules. The hydrophobic beta-sheet faces of apoptin
molecules will form binding interactions and the
hydrophilic/charged beta-sheet faces of apoptin molecules will form
binding interactions. It appears that the formation of amyloid-like
structure resulting in approximately 30 mers is an intrinsic
capacity of apoptin related to its tumor-specific
apoptosis-inducing activity in transformed and aberrant cells.
[0031] In an earlier application (WO02/079222, Fusion proteins for
specific treatment of cancer and auto-immune diseases), a
polypeptide complex is disclosed with apoptosis-inducing activity
and a viral vector comprising the nucleic acid encoding this
polypeptide that achieves the goal of (specifically) killing
aberrant cells, e.g., tumor cells, by targeting these cells and, as
a result, specifically inducing apoptosis in these tumor cells. It
is believed that this eradication of aberrant cells is the result
of uptake of the polypeptide or of the viral vector bearing the
nucleic acid encoding this polypeptide bearing apoptosis-inducing
activity, by both aberrant cells and non-transformed healthy cells,
followed by selective induction of apoptosis in the aberrant cells
only, leaving the healthy cells basically unaltered.
SUMMARY OF THE DISCLOSURE
[0032] Provided is a proteinaceous molecule comprising at least a
domain that comprises an amino acid sequence that specifically
binds to an MHC-peptide complex functionally connected with a
substance that induces apoptosis in aberrant cells, but not normal
cells. In a second embodiment, the substance in the proteinaceous
molecule hereof is an apoptosis-inducing polypeptide or protein. In
yet another embodiment, the apoptosis-inducing polypeptide or
protein and the domain are linked via peptide bonds. In a further
embodiment, the apoptosis-inducing polypeptide or protein and the
domain comprise a single polypeptide chain. In a preferred
embodiment, a proteinaceous molecule is provided wherein the domain
specifically binds an MHC-1-peptide complex. In another embodiment,
provided is a proteinaceous molecule comprising at least a domain
that comprises an amino acid sequence that specifically binds to an
MHC-peptide complex functionally connected with a substance that
induces apoptosis in aberrant cells, but not normal cells, wherein
the peptide within the MHC-peptide complex comprises a MAGE
peptide. In a further embodiment, the proteinaceous molecule
comprises a substance that induces apoptosis is provided, wherein
the substance is apoptin or a fragment and/or derivative thereof,
being capable of inducing apoptosis in aberrant cells, but not
normal cells. In one embodiment, the proteinaceous molecule
comprises the substance in which the substance is a statin. In
another embodiment, provided is a proteinaceous molecule comprising
at least a domain that comprises an amino acid sequence that
specifically binds to an MHC-peptide complex functionally connected
with a substance that induces apoptosis in aberrant cells, but not
normal cells, wherein the domain is linked to the substance through
a non-peptidic bond.
[0033] It is a goal of the disclosure to address the above-listed
limitations related to specificity of apoptosis-inducing activity
toward cancer cells. A second goal is to provide a pharmaceutically
active molecule that specifically and effectively induces apoptosis
and that, at the same time, is manufactured in a less cumbersome
manner. In particular, it is a goal of the present invention to
specifically and selectively target aberrant cells and induce
apoptosis of these aberrant cells, leaving healthy cells
essentially unaffected. MHC-1-peptide complexes on tumors of almost
any origin are valuable targets, whereas MHC-2-peptide complexes
are valuable targets on tumors of hematopoietic origin.
[0034] Thus, provided is a polypeptide comprising a binding domain
specifically binding to a certain MHC-p complex exposed on the
surface of an aberrant cell and a polypeptide specifically inducing
apoptosis (programmed cell death) in this aberrant cell.
Preferably, the binding domain and the polypeptide in the fused
polypeptide are separated by a linker amino acid sequence.
Typically herein, a single polypeptide comprising the necessary
binding domain and the necessary apoptosis-inducing polypeptide
separated by an amino acid sequence is provided. This does not mean
that every molecule hereof may only consist of a single polypeptide
chain. It is, e.g., possible to provide one or more connected
binding domains for another polypeptide chain on the polypeptide
hereof comprising the binding domain and the apoptosis-inducing
polypeptide. The third polypeptide would typically not comprise one
or more coupled copies of an antibody binding domain and/or an
apoptosis-inducing domain like the binding domain and the
apoptosis-inducing polypeptide. The third polypeptide would be a
polypeptide/protein conferring other desirable properties on the
binding and apoptosis-inducing polypeptide, such as improved
half-life. As an example, the addition of human serum albumin (HSA)
on the polypeptide of the invention may be useful for extension of
half-life, etc.
[0035] Thus, in one embodiment, a proteinaceous molecule is
provided comprising at least a binding domain specific for an
MHC-peptide complex functionally connected with a substance that
induces apoptosis in aberrant cells, but not normal cells.
Preferably, the one or more binding domains and the substance are
functionally connected to each other via peptide bonds between
amino-acid residues flanking the binding domain(s) and flanking the
substance, providing a linear single-chain proteinaceous molecule.
It is also part hereof that the one, two, three and, more
preferably, four, five, six or more binding domains are linked to
the substance via bonds and/or binding interactions other than
covalent peptide bonds between amino acid residues in a linear
sequence. Alternative methods for linking proteinaceous molecules
to each other are numerous and well known to those skilled in the
art of protein linkage chemistry. Protein linkage chemistry not
based on peptide bonds in a single-chain amino acid sequence can be
based on covalent interactions and/or on non-covalent
interactions.
[0036] Not intending to be bound to theory, it appears that the
aberrant cell-specific apoptosis-inducing activity of the
polypeptide hereof results from the specific binding of this
polypeptide to surface exposed antigens on aberrant cells. The
binding domain of the polypeptide hereof recognizes the complex of
MHC-1 loaded with the relevant antigenic peptide present on the
targeted aberrant cell exposing the MHC-p complex. The invention
is, however, equally applicable with MHC-2. In several occasions,
the MHC-p complex is not uniquely exposed by aberrant cells, though
predominantly exposed by aberrant cells. It is part hereof that the
binding domain of the polypeptide hereof recognizes a selected
MHC-1-p complex that is predominantly exposed by the targeted
aberrant cell.
[0037] Many binding domains able to specifically bind to MHC-p
complexes are well known to people of skill in the art. Immediately
apparent are binding domains derived from the immune system, such
as TCR domains and immunoglobulin (Ig) domains. Preferably, the
domains encompass 100 to 150 amino acid residues. Preferably, the
binding domains used herein are similar to variable domains
(V.sub.H or V.sub.L) of antibodies. A good source for such binding
domains are phage display libraries. Whether the binding domain of
choice is actually physically selected from a library or whether
only the information (sequence) is used, is of little relevance. It
is part hereof that the polypeptide preferably encompasses one,
two, three or more variable domains of antibodies ("multivalency"),
linked through peptide bonds with suitable linker sequences.
Classical formats of antibodies such as Fab, whole IgG and
single-chain Fv (linked with, e.g., apoptin) against MHC-peptide
complexes are also within the scope of the invention.
[0038] More and more proteins with apoptosis-inducing activity
specific for aberrant cells become known in the art. As part
hereof, proteins with apoptosis-inducing activity originating from
oncolytic viruses or from other sources can be selected.
Preferably, the 121-amino-acid residue apoptin from CAV is used
herein.
[0039] The techniques of connecting one or multiple connected
binding domains with an apoptosis-inducing polypeptide into a
single molecule or polypeptide are many and well known.
[0040] The single binding domain or the multiple binding domains
and the apoptosis-inducing polypeptide on the polypeptide are
typically separated by a linker sequence. In many instances, a
simple Gly-Ser linker of 4 to 15 amino-acid residues may suffice,
but if greater flexibility of the amino-acid chain is desired,
longer or more complex linkers may be used. Preferred linkers are
(Gly.sub.4Ser).sub.n (SEQ ID NO:4), (GSTSGS)n (SEQ ID NO:5),
GSTSGSGKPGSGEGSTKG (SEQ ID NO:6), EFAKTTAPSVYPLAPVLESSGSG (SEQ ID
NO:7), or any other linker that provides flexibility for protein
folding, stability against protease and flexibility for the
polypeptide to exhibit its dual activity, i.e., specific binding to
aberrant cells and subsequently specifically inducing apoptosis of
the targeted aberrant cells after uptake of at least the
apoptosis-inducing polypeptide of the polypeptide. Another group of
preferred linkers are linkers based on hinge regions of
immunoglobulins. These linkers tend to be quite flexible and quite
resistant to proteases. Examples are given in the experimental
part. The most preferred linkers are EPKSCDKTHT (SEQ ID NO:8)
(IgG1), ELKTPLGDTTHT (SEQ ID NO:9) (IgG3), and ESKYGPP (SEQ ID
NO:10) (IgG4). The binding domain(s) and the apoptosis-inducing
polypeptide may be separated only by a linker. Alternatively, other
useful amino-acid sequences may be introduced between the binding
domain(s) and/or between the binding domain(s) and the
apoptosis-inducing polypeptide, and/or at the N-terminus and/or at
the C-terminus of the polypeptide of the invention.
[0041] As stated before, the binding domains selected according to
the invention are preferably based on, or derived from, an Ig
domain (or a comparable TCR domain or another binding protein). The
Ig domain should have at least one complementarity-determining
region (CDR)-like domain or amino-acid sequence, however,
preferably three. These CDR-like domains or amino-acid sequences
should be separated by framework domains that present the CDR-like
stretches in a proper manner. A suitable domain is a V.sub.H domain
of a human antibody.
[0042] The human V.sub.H domains generally need improvement
regarding their affinity and stability, especially when they are
derived from Fab or ScFv phage libraries. Thus, solubility
engineering steps that transform human V.sub.H domains into soluble
non-aggregating, functional entities are part of the present
invention. The human V.sub.H domain may be "camelized," meaning
that a number of amino-acid residues has been replaced by
amino-acid residues from camelids, such as is present in the llama
Vhh domain. Preferred substitutions are Glu6Ala, Ala33Cys,
Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly, Ser74Ala, Arg83Lys,
Ala84Pro, Trp103Arg or Leu108Gln. Amongst other improvements,
introduction of these preferred amino-acid residue substitutions in
the human Vh sequence improves the solubility and improves the
capability to reverse thermal denaturation. Thus, provided is a
polypeptide hereof, wherein the specific binding domains comprise
an Ig fragment. The origin or the method of selection, as well as
the method of production, of the Ig fragment to be used in the
polypeptide is not really relevant. According to one embodiment, a
polypeptide comprises an Ig fragment, which is a natural, mutated
and/or synthetic VH.
[0043] Although the disclosure contemplates many different
combinations of MEW and antigenic peptides, the most preferred is
the combination of MHC-1 and an antigenic peptide from a
tumor-related antigen presented by MHC-1. Because of HLA
restrictions, there are many combinations of MHC-1-p complexes, as
well as of MHC-2-p complexes, that can be designed based on the
rules for presentation of peptides in MHC. These rules include size
limits on peptides that can be presented in the context of MHC,
restriction sites that need to be present for processing of the
antigen in the cell, anchor sites that need to be present on the
peptide to be presented, etc. The exact rules differ for the
different HLA classes and for the different MHC classes. It is
found that MAGE-derived peptides are very suitable for presentation
in an MHC context. An MHC-1-presentable antigenic peptide with the
sequence Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:11) in MAGE-A was identified,
that is present in almost every MAGE-A variant and that will be
presented by one of the most prevalent MHC-1 alleles in the
Caucasian population (namely HLA-A0201). A second MAGE peptide that
is presented by another MHC-1 allele (namely HLA-CW7) and that is
present in many MAGE variants, like, for example, MAGE-A2, -A3, -A6
and -A12, is E-G-D-C-A-P-E-E-K (SEQ ID NO:12). These two
combinations of MHC-1 and MAGE peptides together could cover 80% of
the Caucasian population. It has been shown in vitro that tumor
cell lines with the correct HLA alleles present are efficiently
killed when the MHC-1-p complex is targeted by a hexavalent complex
of VH domain non-covalent multimers specific for this MHC-1-p
complex (see international publication WO2007/073147). The same
approach can be followed for other MHC molecules, other HLA
restrictions and other antigenic peptides derived from
tumor-associated antigens. Relevant is that the chosen antigenic
peptide to elicit the response must be presented in the context of
an MHC molecule and recognized in that context only. Furthermore,
the antigenic peptide must be derived from a sufficiently
tumor-specific antigen and the HLA restriction must occur in a
relevant part of the population. One of the important advantages of
the present invention is that tumors that down-regulate their
targeted MHC-peptide complex, can be treated with a second binding
molecule comprising at least one binding domain binding to a
different MHC-peptide complex based on the same antigen. If this
one is down-regulated, a third one will be available. Six different
targets on MHC may be available. Since cells need to be "inspected"
by the immune system from time to time, escape through
down-regulation of all MHC molecules does not seem a viable escape
route. In the case that MAGE is the antigen from which the peptide
is derived, escape through down-regulation of the antigen is also
not likely, because MAGE seems important for survival of the tumor
(L. Marcar et al., Cancer Res. 2010, 70:10362-10370). Thus, the
present invention, in an important aspect, reduces or even prevents
escape of the tumor from the therapy, in the sense that the tumor
remains treatable.
[0044] Because one embodiment uses MHC molecules as a target and
individuals differ in the availability of MHC targets, also
provided is a so-called companion diagnostic to determine the HLA
composition of an individual. Although the disclosure preferably
uses a more or less universal (MAGE) peptide, it also provides a
diagnostic for determining the expression of the particular antigen
by the tumor. In this manner, the therapy can be geared to the
patient, particularly also in the set-up to prevent escape as
described hereinbefore. It is known that the HLA restriction
patterns of the Asian population and the black population are
different from the Caucasian population. For these populations,
different MHC-peptide complexes can be targeted, as described in
the detailed description.
[0045] Although the present specification presents more specific
disclosure on tumors, it must be understood that other aberrant
cells can also be targeted by the polypeptides of the present
invention. These other aberrant cells are typically cells that also
proliferate without sufficient control. This occurs in autoimmune
diseases. It is typical that these cells start to show expression
of tumor antigens. In particular, MAGE polypeptides have been
identified in Rheumatoid Arthritis (D. K. McCurdy et al., J.
Rheumatol. 2002, 29:2219-2224). Thus, provided in a preferred
embodiment, a polypeptide wherein the specific binding domain is
capable of binding to an MHC-1-p complex and is covalently bound to
an apoptosis-inducing polypeptide. In a further preferred
embodiment, provided is a polypeptide wherein the specific binding
domain is capable of binding to MHC-1-p complexes comprising an
antigenic peptide derived from a tumor-related antigen, in
particular, MHC-1-p complexes comprising an antigenic peptide
present in a variety of MAGE antigens, covalently bound to an
apoptosis-inducing polypeptide.
[0046] One of the polypeptides exemplified herein has a single
binding domain with the amino-acid sequence, referred to as Vh,
essentially corresponding to: the first 117 amino acids of SEQ ID
NO:2.
[0047] Another one has at least one binding domain comprising the
amino acid sequence:
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSYIS
SDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSPRGYYYYGLDL
WGQGTTVTVSS (SEQ ID NO:13; 11H).
[0048] One of the polypeptides exemplified herein has two binding
domains with the amino-acid sequence, referred to as (Vh).sub.2,
essentially corresponding to:
MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGS
NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLV TVSS (SEQ
ID NO:14)--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRL
SCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15), with, for
example, the linker amino-acid sequence GGGGSGGGGS (SEQ ID NO:16)
and two AH5 Vh binding domains.
[0049] One of the polypeptides exemplified herein has three binding
domains with the amino-acid sequence, referred to as (Vh).sub.3,
essentially corresponding to:
MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGS
NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLV TVSS (SEQ
ID NO:14)--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRLSCAAS
GFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15)--linker
amino-acid
sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAV
ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYW
GQGTLVTVSS (SEQ ID NO:15), with, for example, the linker amino-acid
sequences GGGGSGGGGS (SEQ ID NO:16) and three AH5 Vh binding
domains.
[0050] One of the polypeptides exemplified herein has four binding
domains with the amino-acid sequence, referred to as (Vh).sub.4,
essentially corresponding to:
MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGS
NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLV TVSS (SEQ
ID NO:14)--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRLSCAAS
GFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15)--linker
amino-acid
sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGV
AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDY
WGQGTLVTVSS (SEQ ID NO:15)--linker amino-acid sequence--QLQLQESGGG
VVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRF
TISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15),
with, for example, the linker amino-acid sequences GGGGSGGGGS (SEQ
ID NO:16) and four AH5 Vh binding domains.
[0051] One of the polypeptides exemplified herein has an
apoptosis-inducing polypeptide with the amino-acid sequence,
referred to as apoptin, essentially corresponding to:
MNALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSLCGCANARAPTLRSATA
DNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTAKRRIRL (SEQ ID
NO:3).
[0052] Preferred polypeptides according to the invention have an
amino-acid sequence, referred to as Vh-apoptin, essentially
corresponding to MAQLQLQESGGGVVQPGRSLRL
SCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:14)--linker
amino-acid sequence--NALQEDTPPGPSTVFRPPTSS
RPLETPHCREIRIGIAGITITLSLCGC
ANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPS
RPRTAKRRIRL (SEQ ID NO:17), referred to as (Vh).sub.1-apoptin or
AH5-apoptin, with, for example, the linker amino-acid sequences
GGGGSGGGGS (SEQ ID NO:16) and one AH5 Vh binding domain or to
MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGM
HWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA
VYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:14)--linker amino-acid
sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTV SS (SEQ
ID NO:15)--linker amino-acid sequence--NALQEDTPPGPSTVFRPPTSS
RPLETPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPP
SKKRSCDPSEYRVSELKESLITTTPSRPRTAKRRIRL (SEQ ID NO:17), referred to
as (Vh).sub.2-apoptin, with, for example, the linker amino-acid
sequences GGGGSGGGGS (SEQ ID NO:16) and two AH5 Vh binding domains
or to MAQLQLQESGGGVVQPGRSLRLSCAAS
GFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:14)--linker
amino-acid
sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVA
VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDY
WGQGTLVTVSS (SEQ ID NO:15)--linker amino-acid
sequence--QLQLQESGGGVV
QPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTIS
RDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID
NO:15)--linker amino-acid
sequence--NALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAG
ITITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSEL
KESLITTTPSRPRTAKRRIRL (SEQ ID NO:17), referred to as
(Vh).sub.3-apoptin, with, for example, the linker amino-acid
sequences GGGGSGGGGS (SEQ ID NO:16) and three AH5 Vh binding
domains or to MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMH
WVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:14)--linker amino-acid
sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTV SS (SEQ
ID NO:15)--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRLS
CAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTL
YLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15)--linker
amino-acid sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK
EREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSY
YVPDYWGQGTLVTVSS (SEQ ID NO:15)--linker amino-acid sequence--NALQED
TPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGF
KNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTAKRRIRL (SEQ ID NO:17),
referred to as (Vh).sub.4-apoptin, with, for example, the linker
amino-acid sequences GGGGSGGGGS (SEQ ID NO:16) and four AH5 Vh
binding domains.
[0053] Preferred polypeptides according to the invention have an
amino-acid sequence including a cathepsin-L cleavage site
(RKELVTPARDFGHFGLS) (SEQ ID NO:18), referred to as Vh-cath-apoptin,
essentially corresponding to: MAQLQLQESGGGVVQPGRSLRLSC
AASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:14)--linker
amino-acid sequence--RKELVTPARDFGHFGLSNALQEDTPPGPSTVFRPPTSSRPLETPH
CREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCD
PSEYRVSELKESLITTTPSRPRTAKRRIRL (SEQ ID NO:19), referred to as
(Vh).sub.1-cath-apoptin, with, for example, the linker amino-acid
sequences GGGGSGGGGS (SEQ ID NO:16) and one AH5 Vh binding domain
or to MAQLQLQESGGGVV
QPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTIS
RDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID
NO:14)--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSS
YGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA
EDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15)--linker amino-acid
sequence--RKELVTPARDFGHFGLS
NALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSL
CGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLIT
TTPSRPRTAKRRIRL (SEQ ID NO:19), referred to as
(Vh).sub.2-cath-apoptin, with, for example, the linker amino-acid
sequences GGGGSGGGGS (SEQ ID NO:16) and two AH5 Vh binding domains
or to MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREG
VAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPD
YWGQGTLVTVSS (SEQ ID NO:14) \--linker amino-acid
sequence--QLQLQESGGG
VVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRF
TISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15)
\--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSY
GMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15) \--linker amino-acid
sequence--RKELVTPARDFGHFGLSNALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSL
CGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLIT
TTPSRPRTAKRRIRL (SEQ ID NO:19), referred to as
(Vh).sub.3-cath-apoptin, with, for example, the linker amino-acid
sequences GGGGSGGGGS (SEQ ID NO:16) and three AH5 Vh binding
domains or to MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREG
VAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPD
YWGQGTLVTVSS (SEQ ID NO:14)--linker amino-acid sequence--QLQLQESGGG
VVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRF
TISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID
NO:15)--linker amino-acid sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSS
YGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA
EDTAVYYCAGGSYYVPDYWGQGTLVTVSS (SEQ ID NO:15)--linker amino-acid
sequence--QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGS
NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLV TVSS (SEQ
ID NO:15)--linker amino-acid sequence--RKELVTPARDFGHFGLSNAL
QEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSES
TGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTAKRRIRL (SEQ ID
NO:19), referred to as (Vh).sub.4-cath-apoptin, with, for example,
the linker amino-acid sequences GGGGSGGGGS (SEQ ID NO:16) and four
AH5 Vh binding domains.
[0054] Equally preferred are polypeptides hereof similar to those
listed above, now comprising Vh binding domain 11H instead of AH5.
It is appreciated that additional preferred constructs according to
the invention have other cleavage sites such as, but not limited
to, e.g., the cathepsin-B cleavage site with sequence GFQGVQFAGF
(SEQ ID NO:20). Even more preferred constructs comprising
consecutive binding domains comprise different preferred linker
amino-acid sequences between a first and a second binding domain,
and a second, a third and a fourth binding domain. In the
above-outlined examples of polypeptides, the apoptosis-inducing
polypeptide or protein is positioned at the C-terminal site of the
one or more binding domains. Polypeptides with the
apoptosis-inducing polypeptide or protein, like, for example,
apoptin, positioned at the N-terminal site of the one or more
binding domains are also part hereof. See also FIG. 5 for examples
of preferred molecules hereof.
[0055] The disclosure comprises the nucleic acids encoding the
polypeptides. The molecules can be produced in prokaryotes as well
as eukaryotes (one has to take care because apoptin induces cell
death in cell lines (which are essentially tumor cells)). The codon
usage of prokaryotes may be different from that in eukaryotes. The
nucleic acids can be adapted in these respects. Also, elements that
are necessary for secretion may be added, as well as promoters,
terminators, enhancers, etc. Also, elements that are necessary
and/or beneficial for the isolation and/or purification of the
polypeptides may be added. Typically, the nucleic acids are
provided in an expression vector suitable for the host in which
they are to be produced. Choice of a production platform will
depend on the size of the molecule, the expected issues around
protein folding, whether additional sequences are present that
require glycosylation, expected issues around isolation and/or
purification, etc. Thus, nucleic acids according to the invention
are typically adapted to the production and purification platform
in which the polypeptides according to the invention are to be
produced. Thus, provided is a nucleic acid encoding a polypeptide
according to the disclosure, as well as an expression vector
comprising such a nucleic acid. For stable expression in a
eukaryote, it is preferred that the nucleic acid encoding the
polypeptide be integrated in the host cell genome (at a suitable
site that is not silenced). Thus, the disclosure comprises in a
particular embodiment, a vector comprising means for integrating
the nucleic acid in the genome of a host cell.
[0056] The disclosure further comprises the host cell or the
organism in which the polypeptide-encoding nucleic acid is present
and which is thus capable of producing the polypeptide according to
the invention.
[0057] Included herein are also the methods for producing a
polypeptide hereof, comprising culturing a host cell comprising a
suitable nucleic acid, allowing for expression of the nucleic acid
and harvesting the polypeptide.
[0058] For administration to subjects, the polypeptide is
formulated. Typically, these polypeptides will be given
parenterally. For formulation, simply water (saline) for injection
may suffice. For stability reasons, more complex formulations may
be necessary. The disclosure contemplates lyophilized compositions
as well as liquid compositions, provided with the usual additives.
Thus, provided is a pharmaceutical composition comprising a
polypeptide complex according to the disclosure and suitable
diluents and/or excipients.
[0059] The dosage of the polypeptides according to the invention
must be established through animal studies and clinical studies in
so-called rising-dose experiments. Typically, the doses will be
comparable with present day antibody dosages (at the molar level,
the molecular weight of the molecules may differ from that of
antibodies). Typically, such dosages are 3-15 mg/kg body weight, or
25-1000 mg per dose.
[0060] It has been established in the field of tumor therapy that a
single agent is hardly ever capable of eradication of a tumor from
a patient. Especially in the more difficult to treat tumors, the
first applications of the polypeptides hereof will (at least
initially) probably take place in combination with other treatments
(standard care). Thus, also provided is a pharmaceutical
composition comprising a polypeptide and a conventional cytostatic
and/or tumoricidal agent. Moreover, also provided is a
pharmaceutical composition comprising a polypeptide for use in an
adjuvant treatment of cancer. Additionally, provided is a
pharmaceutical composition comprising a polypeptide for use in a
combination chemotherapy treatment of cancer. Examples of
chemotherapeutical treatments that are combined with the
pharmaceutical composition are etoposide, paclitaxel and
methotrexate.
[0061] The pharmaceutical compositions will typically find their
use in the treatment of cancer, particularly in forms of cancer
where the targets of the preferred single-chain polypeptide (i.e.,
complexes of MHC and MAGE-A antigenic peptides), are presented by
the tumors. Table 1 gives a list of tumors on which these targets
have, for example, been found. It is easy using (a) binding
domain(s) according to the invention to identify tumors that
present the target MHC-p complexes. This can be done in vitro or in
vivo (imaging).
[0062] The term repeat has the same meaning as domain and motif
throughout the specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1: Specific binding of HLA-A0201/multi-MAGE-A specific
phage clones isolated from a large human non-immune antibody Fab
phage library. Individual antibody Fab expressing phages that were
selected against biotinylated HLA-A0201/multi-MAGE-A were analyzed
by ELISA for their capacity to bind the relevant peptide/MHC
complex only. Streptavidin-coated 96-well plates were incubated
with soluble HLA-A0201/multi-MAGE-A (A2/multiMage) or HLA-A0201/JCV
(A2/JC) peptide/MHC complexes (10 .mu.g/ml), washed to remove
non-bound complexes and incubated with individual phage clones.
Non-binding phages were first removed by three washes with
PBS/TWEEN.RTM., followed by incubation with anti-M13 antibody (1
.mu.g/ml, Amersham) for one hour by room temperature. Finally, the
wells were incubated with an HRP-labeled secondary antibody and
bound phages detected.
[0064] FIG. 2: Phages AH5, CB1 and CG1 specifically bind cells
presenting the multi-MAGE-A peptide. Phages AH5, CB1, CG1, BD5 and
BC7 that had shown specific binding in ELISA using the relevant
HLA-A201/multi-MAGE-A complex and an irrelevant HLA-A201 complex
loaded with a JCV peptide were analyzed for their capacity to bind
cells presenting the multi-MAGE-A peptide in HLA-A0201 molecules.
To this end, human B-LCL (BSM) were loaded with multi-MAGE-A
peptide (10 .mu.g in 100 .mu.l PBS) for 30 minutes at 37.degree.
C., followed by incubation with the Fab phages AH5, CB1, CG1, BD5
and BC7 and analyzed by flow-cytometry using anti-phage antibodies
and a fluorescently labeled secondary antibody.
[0065] FIG. 3: Phages expressing HLA-A2/multi-MAGE-A specific Fab
bind tumor cells of distinct histologic origin. Phages AH5, CB1 and
CG1 specific for HLA-A0201/multi-MAGE-A and a positive control
phage specific for HA-0101/MAGE-A1 were used for staining of
distinct tumor cell lines. To this end, the prostate cancer cell
line LNCaP, the multiple myeloma cell line MDN, the melanoma cell
lines MZ2-MEL43 and G43, and the breast cancer cell line MDA-MD157
were incubated with the different phages (30 minutes at 4.degree.
C.); bound phages were then detected by flow cytometry using
anti-phage antibodies and fluorescently labeled secondary
antibodies.
[0066] FIG. 4: Phage AH5 specifically binds HLA-A0201/multiMAGE-A
complexes only. To determine specificity of the phage AH5, an ELISA
was performed using relevant and irrelevant peptide/MHC complexes.
HLA-A0201 with multi-MAGE-A, gp100, JCV and MAGE-C2 peptides, as
well as HLA-A1 with MAGE-A1 peptide, were coated on streptavidin
96-well plates and incubated with phage AH5.
[0067] FIG. 5: Cartoon displaying examples of preferred binding
molecules. Examples are provided of possible numbers of VH domains
and distinct linker sequences for the construction of multi-domain
proteins. In rows a and c, two examples are provided of
proteinaceous molecules of the invention, comprising one or two
binding domains, with the apoptosis-inducing polypeptide or protein
linked at the C-terminal site of the binding domain. In rows b and
d-f, the exemplified preferred proteinaceous molecules of the
invention comprise one, two, three or four consecutive binding
domains, linked through different linkers between consecutive
domains, with the apoptosis-inducing polypeptide or protein linked
at the N-terminal site of the N-terminal binding domain.
[0068] FIG. 6: The antibody-apoptin fusion protein is produced in
SE-1 bacteria. The pStaby 1.2 tetra-AH5-apoptin (SEQ ID NO:21,
amino-acid sequence (Vh)4-cath-apoptin) construct was introduced
into SE-1 Bacteria and grown to OD=0.6 at 30.degree. C. Protein
production was induced by addition of IPTG to a final concentration
of 1 mM and bacteria were grown at 30.degree. C. for 13 hours. Lane
1: total fraction of bacteria producing the antibody-apoptin fusion
protein; lane 2: periplasmic fraction of bacteria; lane 3:
flow-through of affinity purified antibody-apoptin fusion protein;
lane 4: eluted fraction of antibody-apoptin protein.
[0069] FIG. 7: The antibody-apoptin fusion protein induces
apoptosis in cancer cells. Purified antibody-apoptin fusion protein
was incubated for 6 hours with HLA-A0201-positive cell lines
expressing MAGE-A genes (Daju, Mel624 and MDN) and a
HLA-A0201-positive, MAGE-A-negative EBV-transformed B-cell line
(BSM). As a negative control, a periplasmic fraction of non-induced
SE-1 bacteria was used. After incubation, caspase-3 activity was
measured by "Caspa-Glow" assay (according to the manufacturer's
instructions, Promega). As shown, only HLA-A0201/MAGE-A-positive
cells show active caspase-3 activity. MAGE-A-negative cells and
cells incubated with the negative control protein fraction do not
show any signs of apoptosis.
DETAILED DESCRIPTION OF THE INVENTION
[0070] As outlined in the previous application WO2007/073147, the
desired specific and selective killing of aberrant cells via the
apoptosis machinery can be achieved by contacting these cells with
a multivalent protein complex comprising multiple antigen-specific
MHC-restricted TCRs or MHC-restricted antigen-specific antibodies
or antibody domains. The antigen then is expressed by the targeted
aberrant cells and presented in the context of MHC molecules. This
finding then, opened the possibility to selectively kill a
population of cells that are positive for a certain MHC-p complex
of interest. For example, tumor cells expressing HLA class I
molecules in complex with antigenic peptides derived from
tumor-associated antigens (MAGE-A1, -A2, -A3, -A4, -A5, -A6, -A7,
-A8, -A9, -A10, -A11, -A12, -A12, MAGE-B, MAGE-C2, LAGE-1, PRAME,
NY-ESO-1, PAGE, SSX-2, SSX-4, GAGE, TAG-1, TAG-2, and
HERV-K-MEL).
[0071] In addition, as outlined in our earlier application
WO02/079222 (Fusion proteins for specific treatment of cancer and
auto-immune diseases), the desired specific and selective killing
of aberrant cells via the apoptosis machinery can be achieved by
contacting these cells with recombinant apoptosis-inducing apoptin
protein. This specific and selective killing can be achieved in one
of several ways. For example, when apoptin is fused with a
polypeptide such as TAT or PTD4, that adds a signal for cellular
uptake to apoptin; or by micro-injecting targeted aberrant cells
specifically with recombinant apoptin protein; or, for example, by
contacting cells including aberrant cells with non-replicative
viruses bearing the apoptin nucleic acid. Once delivered in a
non-specific manner to aberrant cells, apoptin exposes its
apoptosis-inducing activity specific for transformed and aberrant
cells, such as tumor cells. Overcoming the barrier of providing
predominantly aberrant cells with this ability of apoptin to
trigger their cell-death machinery specifically, efficient and
selectively, would open the possibility to develop new generation
anti-cancer therapeutics acting on aberrant cells only, thus being
able to arrest tumor growth and moreover being able to bring
existing tumors into regression.
[0072] In the current application, selectivity and affinity for
cancer cell-specific antigens were combined with cancer
cell-specific apoptosis-inducing activity in a polypeptide of the
invention. The present invention thus discloses that the goal of
specifically killing aberrant cells can be achieved by providing a
polypeptide comprising a polypeptide domain specifically binding to
a certain antigen associated with aberrant cells, and comprising a
cell death-inducing polypeptide. After uptake of the polypeptide,
these aberrant cells are selectively and specifically killed by the
apoptosis-inducing activity of the polypeptides. Thus, in a first
embodiment, this molecule binds specifically to an antigen unique
to aberrant cells, and thereby transfers its ability to selectively
induce apoptosis into the targeted aberrant cells. The
intracellular delivery of the apoptosis-inducing activity of the
molecules into aberrant cells predominantly leaves healthy cells
and tissue essentially unaltered, even if targeted to a certain
level by the molecules. It is part of the disclosure that the
polypeptide is presented as a monomer or as a non-covalent complex
of monomers.
[0073] The terms protein and polypeptide have roughly the same
meaning throughout the text of this application and refer to a
linear proteinaceous sequence comprising two or more amino-acid
residues. In the context of the proteins, protein domains, and
domains that specifically bind to MHC-p complexes, binding
molecules, binding domains and polypeptides have the same meaning
as proteins.
[0074] The term apoptosis refers to the process of programmed cell
death. The term apoptosis-inducing activity means the ability of a
protein or a virus or any other polypeptide, compound, organism or
molecule according to the current invention, to activate, induce,
influence and/or stimulate the cell death machinery of a cell,
resulting in the process of programmed cell death. An aberrant cell
is defined as a cell that deviates from its usual and healthy
normal counterparts in its abnormal growth characteristics.
[0075] Apoptin bears tumor cell-specific apoptosis-inducing
activity, acts independently of p53 and is, in several tumor cell
types, insensitive to Bcr-Abl and Bcl-xl and even stimulated by
Bcl-2. These characteristics attribute to the high potency of
apoptin when applied in the development of new anti-tumor
medicaments according to the invention.
[0076] The binding domain that specifically recognizes and binds to
an MHC-p complex can be a TCR or a functional fragment thereof
(together herein referred to as TCRs) and/or an antibody that
mimics TCR specificity, for example, a genetically engineered
antibody such as a single-chain variable fragment (scFv) or the
variable domain V of the heavy chain H of an antibody (referred to
throughout the text as VH, Vh or V.sub.H). In the specification,
MHC-peptide complex and MHC-peptide antigen have the same meaning.
In the context of a peptide that is presented by an MHC molecule,
forming an MHC-p complex, the terms peptide, peptidic antigen,
antigenic epitope and antigenic peptide refer to the same peptide
in the MHC-p complex.
[0077] Multivalent TCR domain complexes and therapeutic
applications thereof are known in the art. In application
WO2004/050705, a multivalent TCR domain complex comprising at least
two TCRs, linked by a non-proteinaceous polymer chain or a linker
sequence composed of amino-acid residues, is disclosed. The
disclosed use of the TCR complex is in targeting cell delivery of
therapeutic agents, such as cytotoxic drugs, which can be attached
to the TCR complex. Furthermore, WO2004/050705 focuses on the use
of a multivalent TCR complex for the delivery of a therapeutic
agent, e.g., a toxic moiety for cell killing, to a target cell.
[0078] The specific binding capacity of one or multiple MHC-p
complex binding domain(s) fused with an apoptosis-inducing
polypeptide and rendered with the ability to be taken up
specifically by the targeted aberrant cell of the current invention
is sufficient to induce apoptosis of a target cell expressing the
relevant antigen. Any binding domain capable of specifically
binding to an MHC-p complex, comprising either MHC class I or MHC
class II proteins, is suitably used in an apoptosis-inducing
single-chain polypeptide hereof. Also according to the disclosure,
any proteinaceous molecule capable of specifically inducing
apoptosis in an aberrant cell is suitably used in an
apoptosis-inducing single-chain polypeptide hereof. In one
embodiment, therefore, this molecule comprises one or multiple
polypeptide binding domains connected through regular peptide bonds
comprising an amino acid sequence corresponding to a V.sub.H domain
of a human antibody specifically binding to an MHC-p complex, and a
polypeptide comprising the amino acid sequence corresponding to
apoptin-inducing apoptosis once engulfed by a target cell,
connected through peptide bonds between the V.sub.H domain(s) and
apoptin.
[0079] The terms cancer cell and tumor cell have basically the same
meaning throughout the specification.
[0080] This disclosure is, like in application WO2007/073147,
primarily exemplified by the generation of a single-chain monomeric
polypeptide encompassing one V.sub.H domain or multiple V.sub.H
domains and apoptin, which is specific for a tumor antigen and
which specifically kills tumor cells.
[0081] This single-chain monomeric polypeptide has therapeutic
value in the treatment of cancer and autoimmune diseases. Moreover,
the skilled person will appreciate that it is not limited to any
type of antigen, and that single-chain monomeric polypeptides are
provided that can selectively kill target cells, like, for example,
selected aberrant cells, expressing any antigen, known or still to
be discovered, presented in the context of MHC.
[0082] Preferably, a molecule hereof is capable of specifically and
efficiently recognizing and binding to a cancer-specific epitope or
an epitope associated with autoimmune disorders or an epitope
presented by any other aberrant cell, for all examples in the
context of MHC. Cancer cells may express a group of antigens termed
"cancer testis antigens" (CT). These CT are presented as antigenic
peptides by MHC molecules to CTLs. In fact, these CT are
immunogenic in cancer patients as they may elicit anti-cancer
responses. They exhibit highly tissue-restricted expression and are
considered promising target molecules for cancer vaccines and other
immune intervention strategies.
[0083] To date, more than 44 CT gene families have been identified
and their expression has been studied in numerous cancer types. For
example, bladder cancer, non-small lung cancer, prostate cancer,
melanoma and multiple myeloma express CT genes to a high level.
Experiments have shown that expression of these CT genes was indeed
testis restricted in healthy individuals. Other antigens that were
shown to elicit immune responses in cancer patients include
differentiation antigens such as, for example, the melanoma
antigens gp100, Mart-1, Tyrosinase, or antigens that are
over-expressed in cancer cells, such as, for example, p53,
Her-2/neu, WT-1. In a preferred embodiment, the polypeptide
according to the invention is capable of recognizing and binding to
an MHC class I-p complex or to an MHC class II-p complex with the
antigenic peptide in the MHC-p complex derived from a tumor
antigen, in particular, melanoma-associated antigens, and with the
MHC-p complex specifically expressed at tumor cells, leaving
healthy cells and tissue essentially unaltered. The general benefit
of the disclosure is that, where up until now targets associated
with cell surfaces were the predominant goal, intracellular targets
now become available through presentation by MHC-1 and/or MHC-2.
This means that a renewed survey of intracellular antigens will be
carried out to identify intracellular antigens that are tumor
specific enough to merit using them as targets in the disclosure.
Such a screen has already been carried out in the context of tumor
vaccination schemes. Targets that are valuable (because of
sufficient specificity, not necessarily efficacy) as tumor vaccine
candidates will also be valuable: MAGE-A1, -A2, -A3, -A4, -A5, -A6,
-A7, -A8, -A8, -A10, -A11, -A12, MAGE-B, MAGE-C2, LAGE-1, SSX-2,
SSX-4, PRAME, PAGE, NY-ESO-1, GAGE, and HERV-K-MEL.
[0084] Human tumor antigen-derived antigenic peptides presented by
MHC class II molecules have been described, with nearly all of them
being associated with multiple myeloma or malignant melanoma. The
first melanoma antigenic peptide found was MAGE-1. Furthermore,
three melanoma epitopes were found to originate from the MAGE
family of proteins and presented by HLA-DR11 and HLA-DR13. Another
set of melanoma antigens, known to contain also MHC class I tumor
antigens, comprises Melan-A/MART-1, gp100 and Tyrosinase. For an
overview of T-cell epitopes that are of use for the present
invention, also see the World Wide Web at
cancerimmunity.org/peptidedatabase/Tcellepitopes.htm.
[0085] The first discovered CT, belonging to the group of MAGE-A
antigens, has an expression profile that is uniquely restricted to
cancer cells and testis cells. However, testis cells are not
targeted by the immune system, as they lack expression of MHC
molecules. The MAGE-A antigens belong to a family of twelve genes
that show high homology. Their expression has been associated with
early events in malignant cell transformation and metastatic spread
of cancer cells. In addition, down-regulation of MAGE-A expression
may induce apoptosis in cancer cells. Within the MAGE-A genes,
several antigenic epitopes are known by persons in the art.
Antigenic peptides usually are presented as 8- or 9-mer amino acid
peptides by MHC class I molecules. In addition, epitopes are known
that are present in multiple MAGE-A genes due to the high homology
between the different MAGE-A genes. These epitopes may be
considered as multi-MAGE-A epitopes and are presented on cancer
cells of various histologic origin. Therefore, they might serve as
universal targets for anti-cancer therapy.
[0086] MHC molecules are also important as signal-transducing
molecules, regulating immune responses. Cross-linking of MHC Class
I molecules on B and T cells initiates signals that can result in
either anergy, or apoptosis, or, alternatively, in cell
proliferation and cytokine production. Several intracellular
signaling pathways have been identified that are induced by MHC
class I cross-linking. These include 1) phosphorylation of tyrosine
kinases, leading to enhanced levels of intracellular calcium ions;
2) activation of the JAK/STAT pathway; and 3) inhibition of PI3K,
resulting in the activation of JNK activation. In addition,
cross-linking of MHC Class I/II molecules results in the engulfment
of the MHC-p complexes with bound single-chain polypeptide
according to the invention, allowing the delivery of, e.g., toxic
proteins or toxic compounds.
[0087] A further aspect relates to a method for providing the
molecule hereof. As described hereinabove, it typically involves
providing a nucleic acid construct encoding the desired
polypeptide. The nucleic acid construct can be introduced,
preferably via a plasmid or expression vector, into a prokaryotic
host cell and/or in eukaryotic host cell capable of expressing the
construct. In one embodiment, a method to provide a single-chain
apoptosis-inducing protein comprises the steps of providing a host
cell with one or more nucleic acid(s) encoding the protein, and
allowing the expression of the nucleic acids by the host cell.
[0088] Preferred host cells are bacteria, like, for example,
bacterial strain BL21 or strain SE1, or mammalian host cells, more
preferably human host cells. Suitable mammalian host cells include
human embryonic kidney (HEK-293) cells, PER.C6.RTM. cells or
Chinese hamster ovary (CHO) cells, which can be commercially
obtained. Insect cells, such as S2 or S9 cells, may also be used
using baculovirus or insect cell expression vectors, although they
are less suitable when the polypeptides according to the invention
include elements that involve glycosylation. The single-chain
polypeptides produced can be extracted or isolated from the host
cell or, if they are secreted, from the culture medium of the host
cell. Thus, in one embodiment, a method comprises providing a host
cell with one or more nucleic acid(s) encoding the polypeptides,
allowing the expression of the nucleic acids by the host cell. It
is included that the molecules are capable of specifically and
effectively binding to an MHC-p complex and subsequently inducing
apoptosis after engulfment of the bound molecules by the targeted
aberrant cell. Methods for the recombinant expression of
(mammalian) proteins in a (mammalian) host cell are well known in
the art.
[0089] As will be clear, a molecule hereof finds its use in many
therapeutic applications and non-therapeutic applications, e.g.,
diagnostics or scientific applications. Provided herein is a method
for inducing ex vivo or in vivo apoptosis of a target cell,
comprising contacting the cell with a polypeptide according to the
invention in an amount that is effective to induce apoptosis. The
target cells can be conveniently contacted with the culture medium
of a host cell that is used for the recombinant production of the
polypeptide. In one embodiment, it can be used for in vitro
apoptosis studies, for instance, studies directed at the
elucidation of molecular pathways involved in MHC class I- and
class II-induced apoptosis. Molecules hereof may also be used for
the detection of (circulating) tumor cells.
[0090] Preferably, the single-chain molecule is used for triggering
apoptosis of aberrant cells in a subject, more preferably a human
subject. For therapeutic applications in humans, it is, of course,
preferred that a single-chain molecule does not contain amino-acid
sequences of non-mammalian origin. More preferred are single-chain
proteins, which only contain human amino-acid sequences apart from,
e.g., apoptin, or which contain human amino-acid sequences
including a minimal number of camelid-derived amino-acid residues.
Therefore, a therapeutically effective amount of a polypeptide
binding to a disease-specific epitope can be administered to a
patient to stimulate specific apoptosis of aberrant cells without
affecting the viability of (normal) cells not expressing the
disease-specific epitope. It is demonstrated herein that a method
of the invention allows for the killing of cells in an
antigen-specific, MHC-restricted fashion. In a specific embodiment,
the disease-specific epitope is a cancer-specific epitope, for
example, a melanoma-specific epitope. The killing of aberrant
cells, while minimizing or even totally avoiding the death of
normal cells, will generally improve the therapeutic outcome of a
patient following administration of the single-chain polypeptides
according to the invention.
[0091] Accordingly, there is also provided a polypeptide according
to the invention as a medicament. In another aspect, provided is
the use of a polypeptide for the manufacture of a medicament for
the treatment of cancer, autoimmune disease or any other disease of
which the symptoms are reduced upon killing the cells expressing a
disease-specific antigenic peptide or epitope in the context of
MHC. For example, a polypeptide according to the invention is
advantageously used for the manufacture of a medicament for the
treatment of melanoma.
[0092] Antibody fragments of human origin can be isolated from
large antibody repertoires displayed by phages. One aspect of the
invention is the use of human antibody phage display libraries for
the selection of human Fab or human VhCh fragments specific for MHC
class I molecules presenting cancer testis antigenic peptides.
Antibody fragments specific for MHC class I, i.e., HLA-A0201
molecules presenting a multi-MAGE-A epitope, have been selected
(essentially as described in R. A. Willemsen et al., Cytometry A,
2008, 73:1093-1099) and shown to bind the relevant antigen only. As
these antibody Fab fragments usually display low affinity, a method
is provided that allows the generation of high avidity antibody
chains able to induce apoptosis in a MHC-restricted antigenic
peptide-specific way. An aspect of the present invention is the
development of a single-chain polypeptide comprising multiple (up
to four) antigen binding domains to enhance MHC-p complex binding
avidity. Enhancing MHC-p complex binding avidity results in
efficient cross-linking of the MHC-p complexes and engulfment of
the MHC-p complexes with bound single-chain polypeptides according
to the invention, subsequently followed by apoptin-mediated
induction of apoptosis.
[0093] Throughout the specification, the term fragment refers to an
amino-acid sequence that is part of a protein domain or that builds
up an intact protein domain. Fragments according to the invention
must have binding specificity for the respective target.
[0094] An MHC-p complex-specific polypeptide in a monovalent or
multivalent single-chain polypeptide form of the invention is, for
example, an MHC-restricted antigen-specific TCR-like antibody (Ab)
or functional fragment thereof, which is used as a monomer or which
is multimerized at the DNA level in order to obtain a single-chain
polypeptide construct upon expression.
[0095] Antibody Fab fragments are composed of antibody variable
domains, responsible for antigen binding, and parts of the constant
domains, lacking immunologic function. The variable domains in
antibody Fab fragments, the variable heavy (V.sub.H) and variable
light (V.sub.L) chain domains both bind the antigen. However, in
many circumstances, the V.sub.H chain alone is able and sufficient
to bind antigen, for example, in VhCh fragments. As such, antibody
V.sub.H domains would provide small functional binding units.
[0096] Human V.sub.H domains usually do not meet the standards for
stability and efficient expression that are required by the field,
especially when derived from Fab and ScFv libraries. They tend to
be unstable and poorly expressed. A process called "camelization"
may be used to convert human V.sub.H into more stable antibody
fragments.
[0097] The human antibody germline region V.sub.H-3 displays high
homology with antibody V.sub.H fragments of llamas. Llamas have two
types of antibodies, those composed of heavy and light chains, and
antibodies that only contain heavy chains. These heavy-chain only
antibodies bind antigens similar to classical antibodies composed
of heavy and light chains. The smallest functional llama antibody
binding domain, the V.sub.HH domain, also called single domain
antibodies (sdAb), have been shown to be expressed well and may
bind antigen with high affinity. In addition, it has been shown
that some of the characteristics, such as ease of expression and
stability, of llama sdAb can be transferred to, e.g., human V.sub.H
by replacing a few amino acids in the human V.sub.H for those of
llama V.sub.H. High avidity antibody molecules can then be
generated by ligation of several "camelized" human VH domains into
one single molecule.
[0098] Preferred molecules comprise 1-6 "camelized" or
"non-camelized" human VH domains interspersed by short linkers
providing flexibility between the VH domains and between the
binding domains and apoptin. For example, a tetravalent protein is
generated that is specific for the HLA-A0201 restricted
multi-MAGE-A epitope as part of a single-chain polypeptide
comprising the apoptin polypeptide. These proteins according to the
invention are referred to as a single-chain protein or
(single-chain) polypeptide or monomeric protein or monomeric
polypeptide. See, for further details, the outlined Examples below.
It is to be appreciated that this technology allows for the
generation of multivalent single-chain proteins that comprise any
number of the same or different binding domains such as single
domain antibodies or human VH domains. For several reasons (such as
ease of production), repeats are not always the best option. Thus,
the invention also contemplates using different binding domains
(essentially recognizing the same target) separated by several
different linkers, as shown in FIG. 5.
[0099] For example, a tetravalent single-chain polypeptide
according to the invention, consisting of four linked camelized or
non-camelized human VH domains connected through peptide bonds to
apoptin, is used, for example, to induce apoptosis in cancer cells
that express both the MAGE-A genes and HA-A0201. Noteworthy,
specificity for this MHC-p complex is provided in this way as cells
that do not express HLA-A0201 or that do not express MAGE-A are not
killed. See the Examples section for further details.
[0100] Apoptosis in cancer cells is, for example, detected in vitro
by several assays known to the art, including cytotoxicity assays,
tunnel assays and assays detecting active caspases. In animal
studies, apoptosis is, for example, revealed by monitoring reduced
tumor growth, detection of active caspases or performing a tunnel
assay on isolated tumor material.
[0101] In literature, it is shown that a single nine amino-acid
(A.A.) peptide present in MAGE-A2, -A3, -A4, -A6, -A10, and -A12 is
presented by HLA-A0201 on tumor cells, and can be recognized by
cytotoxic T lymphocytes..sup.(1) This nine A.A. peptide with
sequence Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:11) is almost identical to
the HLA-A0201 presented MAGE-A1 peptide Y-L-E-Y-R-Q-V-P-D (SEQ ID
NO:22), except for the anchor residue at position 9. Replacement of
the anchor residue with Valine results in a 9 A.A. peptide with
enhanced binding capacity to HLA-A0201 molecules..sup.(1) Human and
mouse T lymphocytes recognizing the Y-L-E-Y-R-Q-V-P-V (SEQ ID
NO:23) peptide presented by HLA-0201 also recognize the original
MAGE-A Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:11) and Y-L-E-Y-R-Q-V-P-D (SEQ
ID NO:22) peptides presented on tumors of distinct origin. As
diverse tumors may each express at least one MAGE-A gene, targeting
of this so-called multi-MAGE-A epitope includes the vast majority
of tumors. As an example, MAGE-A expression in human prostate tumor
cell lines and in human xenographs was analyzed and shown to be
highly diverse, but in each individual sample tested, at least one
MAGE-A gene was expressed (Table 2), confirming that targeting this
multi-MAGE-A epitope serves as a universal HLA-A0201-restricted
target for therapy. Of course, several other multi-MAGE-A or
multi-target epitopes may be discovered. In principle, the
invention contemplates combinations of tumor-specific
antigen-derived MHC-presented epitopes in different HLA
restrictions of both MHC-I and MHC-II targeted by monomeric or
multimeric (preferably, n=2-4) binding domains linked to an
apoptosis-inducing polypeptide or protein, to induce apoptosis in
aberrant cells. A number of MHC-MAGE peptide combinations that can
be targeted are IMPKAGLLI (MAGE-A3) (SEQ ID NO:21), and HLA-DP4 or
HLA-DQ6/243-KKLLTQHFVQENYLEY-258 (MAGE-A3) (SEQ ID NO:24). Other
examples of tumor-specific complexes of HLA and antigen peptide are
(N. Renkvist et al., Cancer Immunol. Immunother. (2001) V50:3-15):
HLA A1-MAGE-A1 peptide EADPTGHSY (SEQ ID NO:25), HLA A3-MAGE-A1
SLFRAVITK (SEQ ID NO:26), HLA A24-MAGE-A1 NYKHCFPEI (SEQ ID NO:27),
HLA A28-MAGE-A1 EVYDGREHSA (SEQ ID NO:28), HLA B37-MAGE-A1/A2/A3/A6
REPVTKAEML (SEQ ID NO:29), expressed at aberrant cells related to
melanoma, breast carcinoma, SCLC, sarcoma, NSCLC, colon carcinoma.
Further examples are HLA B53-MAGE-A1 DPARYEFLW (SEQ ID NO:30), HLA
Cw2-MAGE-A1 SAFPTTINF (SEQ ID NO:31), HLA Cw3-MAGE-A1 and HLA
Cw16-MAGE-A1 SAYGEPRKL (SEQ ID NO:32), HLA A2-MAGE A2 KMVELVHFL
(SEQ ID NO:33), HLA A2-MAGE-A2 YLQLVFGIEV (SEQ ID NO:34), HLA
A24-MAGE-A2 EYLQLVFGI (SEQ ID NO:35), HLA-A1-MAGE-A3 EADPIGHLY (SEQ
ID NO:36), HLA A2-MAGE-A3 FLWGPRALV (SEQ ID NO:37), HLA B44-MAGE-A3
MEVDPIGHLY (SEQ ID NO:38), HLA B52-MAGE-A3 WQYFFPVIF (SEQ ID
NO:39), HLA A2-MAGE-A4 GVYDGREHTV (SEQ ID NO:40), HLA A34-MAGE-A6
MVKISGGPR (SEQ ID NO:41), HLA A2-MAGE-A10 GLYDGMEHL (SEQ ID NO:42),
HLA Cw7-MAGE-A12 VRIGHLYIL (SEQ ID NO:43), HLA Cw16-BAGE AARAVFLAL
(SEQ ID NO:44), expressed by, for example, melanoma, bladder
carcinoma, NSCLC, sarcoma, HLA A2-DAM-6/-10 FLWGPRAYA (SEQ ID
NO:45), expressed by, for example, skin tumors, lung carcinoma,
ovarian carcinoma, mammary carcinoma, HLA Cw6-GAGE-1/-2/-8 YRPRPRRY
(SEQ ID NO:46), HLA A29-GAGE-3/-4/-5/-6/-7B YYWPRPRRY (SEQ ID
NO:47), both expressed by, for example, melanoma, leukemia cells,
bladder carcinoma, HLA B13-NA88-A MTQGQHFLQKV (SEQ ID NO:48),
expressed by melanoma, HLA A2-NY-ESO-1 SLLMWITQCFL (SEQ ID NO:49),
HLA A2-NY-ESO-1a SLLMWITQC (SEQ ID NO:50), HLA A2-NY-ESO-1a Art
(SEQ ID NO:51), HLA A31-NY-ESO-1a ASGPGGGAPR (SEQ ID NO:52), the
latter four expressed by, for example, melanoma, sarcoma, B
lymphomas, prostate carcinoma, ovarian carcinoma, bladder
carcinoma.
[0102] In one embodiment, human antibody fragments specific for the
HLA-A0201-presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-V (SEQ ID
NO:23) are identified and isolated from a human Fab phage display
library. The selected human antibody fragments are optimized
regarding their specificity and avidity, and provide the amino-acid
sequences used for the design and production of monovalent,
divalent, trivalent, tetravalent, mono-specific single-chain
polypeptides comprising apoptin and specific for efficient binding
of the HLA-A0201-MAGE-A epitope Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:11),
referred to as mono-AH5-apoptin, di-AH5-apoptin, tri-AH5-apoptin,
tetra-AH5-apoptin. In another embodiment, mono-AH5-apoptin,
di-AH5-apoptin, tri-AH5-apoptin, tetra-AH5-apoptin, is produced
comprising a cathepsin-L or cathepsin-B cleavage amino-acid
sequence, providing mono-AH5-Cath-apoptin, di-AH5-cath-apoptin,
tri-AH5-cath-apoptin, tetra-AH5-cath-apoptin, with essentially the
same or comparable binding characteristics compared to
mono-AH5-apoptin, di-AH5-apoptin, tri-AH5-apoptin,
tetra-AH5-apoptin.
[0103] In one embodiment, for example, the mono-AH5-apoptin,
di-AH5-apoptin, tri-AH5-apoptin, tetra-AH5-apoptin, and/or its
equivalents mono-AH5-Cath-apoptin, di-AH5-cath-apoptin,
tri-AH5-cath-apoptin, tetra-AH5-cath-apoptin are used in the
production of a pharmaceutical composition. In yet another
embodiment, monovalent or multivalent AH5-apoptin construct is used
for the production of a pharmaceutical composition for the
treatment of a disease or a health problem related to the presence
of aberrant cells exposing the epitope comprising the
HLA-A0201-MAGE-A epitope Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:11) complex
for monovalent or multivalent AH5-apoptin, monovalent or
multivalent AH5-cath apoptin. The aberrant cells are, for example,
tumor cells. In a further embodiment, monovalent or multivalent
AH5-apoptin and/or its equivalents monovalent or multivalent
AH5-cath-apoptin is used for the treatment of cancer. In yet
another embodiment, monovalent or multivalent AH5-apoptin and/or
its equivalents is used, for example, for the treatment of prostate
cancer, breast cancer, multiple myelomas or melanomas.
[0104] The invention is exemplified by the Examples below.
Abbreviations Used
[0105] A.A., amino acid; Ab, antibody; ADA, anti-drug antibodies;
AFP, alpha-fetoprotein; APC, antigen-presenting cell; .beta.2-M,
.beta.2-microglobulin; CAV, chicken anemia virus; CD, circular
dichroism; CDR, complementarity-determining region; CEA,
carcino-embryonic antigen; CHO, Chinese hamster ovary; CKII.alpha.,
catalytic subunit of casein kinase II; CT, cancer testis antigens;
CTL, cytotoxic T lymphocyte; DC, dendritic cell; E4orf4, adenovirus
early region 4 open reading frame; EBV, Epstein-Barr virus; ELISA,
enzyme linked immunosorbent assay; HAMLET, human
.alpha.-lactalbumin made lethal to tumor cells; HEK, human
embryonic kidney; HLA, human leukocyte antigen; Ig, immunoglobulin;
i.v., intravenously; kDa, kilo Dalton; MAGE, melanoma-associated
antigen; Mda-7, melanoma differentiation-associated gene-7; MHC,
major histocompatibility complex; MHC-p, MHC-peptide; MVM,
parvovirus minute virus of mice; NS1, parvovirus-H1-derived
non-structural protein 1; PBSM, PBS containing 2% non-fat dry milk;
PTD4, protein transduction domain 4; sc-Fv, single-chain variable
fragment; V.sub.HH or sdAb, single-domain antibodies; TCR, T-cell
receptor; VH, Vh or V.sub.H, variable amino-acid sequence of an
antibody heavy domain; TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand.
EXAMPLES
Example 1
Selection of Human Antibody Fragments Specific for
HLA-A0201/Multi-MAGE-A
[0106] To obtain human antibody fragments specific for the
HLA-A0201-presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-G (SEQ ID
NO:11) or Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:23), a Human Fab phage
display library was constructed according to the procedure
previously described by de Haard et al..sup.(2) and used for
selections essentially as described by Chames et al..sup.(3)
Alternatively, a human VhCh library was constructed and used for
selections. Human Fab/VhCh phages (10.sup.13 colony-forming units)
were first pre-incubated for 1 hour at room temperature in PBS
containing 2% non-fat dry milk (PB SM). In parallel, 200 .mu.l
Streptavidin-coated beads (Dynal.TM.) were equilibrated for 1 hour
in PBSM. For subsequent rounds, 100 .mu.l beads were used. To
deplete for pan-MHC binders, each selection round, 200 nM of
biotinylated MHC class I-peptide (MHC-p) complexes containing an
irrelevant peptide (Sanquin, the Netherlands) were added to the
phages and incubated for 30 minutes under rotation. Equilibrated
beads were added, and the mixture was incubated for 15 minutes
under rotation. Beads were drawn to the side of the tube using
magnetic force. To the depleted phage fraction, subsequently
decreasing amounts of biotinylated MHC-p complexes (200 nM for the
first round, and 20 nM for the second and third rounds) were added
and incubated for 1 hour at room temperature, with continuous
rotation. Simultaneously, a pan-MHC class I binding-soluble Fab
(D3) was added to the phage-MHC-p complex mixture (50, 10, and 5
.mu.g for rounds 1-3, respectively). Equilibrated
streptavidin-coated beads were added, and the mixture was incubated
for 15 minutes under rotation. Phages were selected by magnetic
force. Non-bound phages were removed by five washing steps with
PBSM, five steps with PBS containing 0.1% TWEEN.RTM., and five
steps with PBS. Phages were eluted from the beads by 10 minutes
incubation with 500 .mu.l freshly prepared tri-ethylamine (100 mM).
The pH of the solution was neutralized by the addition of 500 .mu.l
M Tris (pH 7.5). The eluted phages were incubated with logarithmic
growing E. Coli TG1 cells (OD.sub.600nm of 0.5) for 30 minutes at
37.degree. C. Bacteria were grown overnight on 2.times. TYAG
plates. Next day, colonies were harvested, and a 10 .mu.l inoculum
was used in 50 ml 2.times. TYAG. Cells were grown until an
OD.sub.600nm of 0.5, and 5 ml of this suspension was infected with
M13k07 helper phage (5.times.10.sup.11 colony-forming units). After
30 minutes incubation at 37.degree. C., the cells were centrifuged,
resuspended in 25 ml 2.times. TYAK, and grown overnight at
30.degree. C. Phages were collected from the culture supernatant as
described previously, and were used for the next round panning.
After three selection rounds, a 261-fold enrichment of Fab phages
was obtained, and 46 out of 282 analyzed clones were shown to be
specific for the HLA-A2-multi-MAGE-A complex (FIG. 1). ELISA using
the HLA-A0201/multi-MAGE-A complexes as well as HLA-A0201 complexes
with a peptide derived from JC virus was used to determine the
specificity of the selected Fab.
Human Fab Specific for the HLA-A0201/Multi-MAGE-A Epitope Bind
Antigen-Positive Cells
[0107] Selected Fab phages were then analyzed for their capacity to
bind HLA-A0201-positive EBV-transformed B-LCL loaded with the
multi-MAGE-A peptide Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:23). The B-LCL
line BSM (0.5.times.10.sup.6) was loaded with multi-MAGE-A peptide
(10 .mu.g in 100 .mu.l PBS) for 30 minutes at 37.degree. C.,
followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and
BC7 and analyzed by flow-cytometry. As shown in FIG. 2, Fab AH5,
CB1 and CG1 specifically bound to the peptide-loaded cells only,
whereas Fab BD5 and BC7 displayed non-specific binding to BSM that
was not loaded with the multi-MAGE-A peptide. No binding was
observed by AH5, CB1 and CG1 to non-peptide-loaded cells.
[0108] Phages presenting AH5, CB1 and CG1, as well as the
HLA-A0101/MAGE-A1-specific Fab phage G8.sup.(4) were then used to
stain tumor cell lines of distinct histologic origin. To this end,
prostate cancer cells (LNCaP), multiple myeloma cells (MDN),
melanoma cells (MZ2-MEL43 and G43), and breast cancer cells
(MDA-MB157) were stained and analyzed by flow cytometry (FIG. 3).
The Fab AH5 specifically bound multiple myeloma cells MDN, and not
the HLA-A0201-negative melanoma and breast cancer cells. Both CB1
and CG1 displayed non-specific binding on the melanoma cell line
G43. The positive control Fab G8 demonstrated binding to all cell
lines tested.
Fab AH5 Binds HLA-A0201/Multi-MAGE-A Complexes Only
[0109] ELISA using multiple peptide/MHC complexes then confirmed
the specificity of Fab-AH5. To this end, HLA-A0201
complexes-presenting peptides multi-MAGE-A, gp100, JCV and MAGE-C2,
as well as a HLA-A1/MAGE-A1 complex, were immobilized on 96-well
plates and incubated with phages displaying Fab AH5 and control Fab
G8. As shown in FIG. 4, AH5 only binds HLA-A0201/multi-MAGE-A and
not the irrelevant complexes HLA-A0201/gp100, HLA-A0201/MAGE-C2,
HLA-A0201/JCV and HLA-A0101/MAGE-A1. The positive control Fab G8
only binds to its relevant target HLA-A0101/MAGE-A1.
Example 2
Production of Monovalent and Multivalent AH5-Apoptin Polypeptides
and Monovalent and Multivalent AH5-Cath-Apoptin Polypeptides
Design of Genes for Production of Tetrameric AH5 VH-Apoptin and AH5
Vh-Cath-Apoptin
[0110] Human antibody germline gene VH3 demonstrates high homology
to llama single domains VHH. Exchange of amino-acids 44, 45 and 47
in the human VH3 genes by amino-acids present in llama VHH at these
positions has shown to enhance stability and expression of the
human VH3 genes. All substitutions described to have an effect on
protein stability and/or solubility include: E6A, A33C, V37F, G44E,
L45R, W47G, S74A, R83K, A84P or L108Q.
[0111] The AH5 VH demonstrates a low homology to germline gene
VH3-33*01 (71% as determined by IMGT homology search); however, its
expression and stability might benefit from the exchange of
amino-acids 6, 44, 45 and 47 and 108 by llama VHH amino-acid
residues, a process called camelization. In addition, a gene was
compiled that upon expression, comprises four AH5 VH domains. To
this end, a gene called tetra-AH5 was designed comprising the pelB
secretion signal, four codon-optimized, camelized AH5 VH domains
with Gly-Ser linkers between each AH5 VH domain, and finally the
apoptin gene (see tetra-AH5-apoptin, see SEQ ID NO:16 for the
amino-acid sequence). The Tetra AH5-cath-apoptin gene comprises the
pelB secretion signal, four codon-optimized, camelized AH5 VH
domains with Gly-Ser linkers between each AH5 VH domain, the
cathepsin-L cleavage site and finally the apoptin gene (see
tetra-AH5-cath-apoptin, see SEQ ID NO:21 for the amino-acid
sequence). This gene was synthesized by "Geneart" (Regensburg,
Germany) and cloned into the pStaby 1.2 vector (Delphi Genetics,
Belgium) for expression in E. coli.
Production and Purification of Tetrameric AH5 VH-Apoptin
Protein
[0112] For expression of tetra-AH5-cath-apoptin, the
pStaby-tetra-AH5-cath-apoptin vector was introduced via
electroporation into SE1 bacteria. Positive clones were grown in
the presence of 2% glucose at 30.degree. C. until OD.sub.600=0.8.
Bacterial TYAG medium was then replaced with TY medium containing 1
mM IPTG to induce expression. After 4 hours or overnight culture at
30.degree. C., bacteria and medium were harvested. The periplasmic
fraction was collected after incubation of bacteria with
PBS/EDTA/NaCl for 30 minutes on ice. Protein expression was
analyzed by SDS-PAGE. It is shown that tetra-cath-apoptin protein
is secreted into the bacterial periplasm and medium (see FIG.
6).
[0113] Tetra-AH5-cath-apoptin was isolated from media and bacterial
periplasm using Ni-affinity purification. To this end, desalted
periplasmic fractions were purified on Acta-FPLC with His-trap
collum or alternatively incubated with Ni-coupled Sepharose-beads
and incubated overnight while stirring gently at 4.degree. C. To
obtain intracellular proteins, bacteria was lysed and cellular
debris removed by centrifugation. After overnight dialysis with
PBS, tetrameric AH5 VH-apoptin and tetrameric AH5-cath-apoptin was
purified with Ni-Sepharose. Purity of the proteins were checked by
SDS-PAGE and protein concentration determined by BCA protein assay
(Pierce).
Example 3
Cell Binding and Internalization of Tetra-AH5-Cath-Apoptin
[0114] Binding capacity of tetra-AH5-cath-apoptin was analyzed by
flow-cytometry. HLA-A0201/multi-MAGE-A-positive tumor cells (Daju,
MDN and mel 624) and HLA-A0201/multi-MAGE-A-negative cells (BSM,
G43 and 293) were incubated on ice with purified protein and
detected by addition of fluorescently labeled anti-His antibodies.
Cells bound by the proteins were quantified and visualized by flow
cytometry. Internalization of tetra-AH5-cath-apoptin was analyzed
by confocal microscopy. To this end, cells were incubated with the
proteins, kept on ice for 30 minutes to allow binding but no
internalization. Next, fluorescently labeled anti-His antibodies
were added. To induce internalization, cells were transferred to
37.degree. C. and fixed with 1% PFA after 5, 10 and 15 minutes.
Example 4
Apoptosis Induction by Tetra AH5-Cath-Apoptin in Diverse Tumor
Cells
Killing of Diverse Tumor Cells by Tetra-AH5-Cath-Apoptin
[0115] Tetra-AH5-cath-apoptin was analyzed for its capacity to
induce apoptosis by incubation with diverse tumor cells, known to
express both HLA-A0201 and MAGE-A genes. The cell lines Daju, Mel
624 (melanoma), PC346C (prostate cancer), and MDN (multiple
myeloma), as well as MAGE-A-negative cells (BSM, and 911, HEK293T),
were incubated with different concentrations of the proteins (in
DMEM medium, supplemented with pen/strep, Glutamine and
non-essential amino acids). Several hours later, cells were
visually inspected for classical signs of apoptosis such as
detachment of the cells from tissue culture plates and membrane
blebbing. It is excepted that the proteins induce apoptosis in the
Daju Mel 624, PC346C and MDN cells. Cells that are not treated with
the proteins will not be affected, as well as cells that do not
express HLA-A0201 (HEK293T) and MAGE-A genes (911 and HEK293T).
Detection of Active Caspase-3
[0116] A classical intra-cellular hallmark for apoptosis is the
presence of active caspase-3.
[0117] To determine whether or not tetra-AHS-cath-apoptin induces
active caspase-3, HLA-A0201/MAGE-A-positive cells (Daju, Mel624 and
MDN), as well as HLA-A0201-positive, but not MAGE-A-negative cells
(BSM), were incubated with tetra-AH5-cath-apoptin. After four and
13 hours, FAM-DEVD-FMK, a fluorescently caspase-3/7 inhibitor, was
added and positively stained cells visualized by fluorescent
microscopy and flow cytometry. It was expected that caspase-3
activity was shown in antigen-positive cells and not in
antigen-negative cells.
Treatment of Tumor-Bearing mice with Tetra-AH5-Apoptin and
Tetra-Cath-Apoptin
[0118] Nude mice (NOD-scid, eight per group) with a palpable
subcutaneous transplantable human tumor (Daju or MDN) was injected
with different doses of tetra-AH5-apoptin or
tetra-AH5-cath-apoptin. As a control, mice were treated with
standard chemotherapy or received an injection with PBS. It was
expected that mice receiving an optimal dose of the proteins would
survive significantly longer that those mice receiving chemotherapy
or PBS.
TABLE-US-00001 TABLE 1 Examples of the frequency of MAGE-A
expression by human cancers. Frequency of expression (%) MAGE-
MAGE- MAGE- MAGE- MAGE- MAGE- MAGE- cancer A1 A2 A3 A4 A6 A10 A11
Melanoma 16 E 36 E 64 E 74 Head and neck 25 42 33 8 N N N Bladder
21 30 35 33 15 N 9 Breast 6 19 10 13 5 N N Colorectal N 5 5 N 5 N N
Lung 21 30 46 11 8 N N Gastric 30 22 57 N N N N Ovarian 55 32 20 E
20 N N osteosarcoma 62 75 62 12 62 N N hepatocarcinoma 68 30 68 N
30 30 30 Renal cell 22 16 76 30 N N N carcinoma E, expressed but
the frequency is not known; N, expression by tumors has never been
determined or observed
TABLE-US-00002 TABLE 1B Expression analysis of MAGE-A1-A6 genes
detected by nested RT-PCR with common primers in squamous cell
carcinoma of the head and neck. Primary site % of positive
expression Larynx 72.7% (8/11) Hypopharynx 100% (2/2) Base of
tongue 50% (1/2) Tonsil 100% (2/2) Total (n = 17) .sup. 76.5%
(13/17) Adapted from: ANTICANCER RESEARCH 26: 1513-1518 (2006)
TABLE-US-00003 TABLE 2 MAGE-A expression in human prostate cancer
cell lines and prostate cancer xenografts. Cell line/ MAGE-
Xenograft A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 LNCaP + ++ ++ ++ +
PC346C + ++ ++ + ++ + + ++ OVCAR + + + + JON ++ ++ ++ + + PNT 2 C2
+ + + + + SD48 + + + + PC-3 + + + PC 374 + PC 346p + ++ ++ ++ + ++
+ PC 82 + + PC 133 ++ + + PC 135 + PC 295 + PC 324 + + + PC 310 +
++ + ++ + PC 339 ++ ++ + ++ + + + Expression of the MAGE-A1, A2,
A3, A4, A5, A6 ,A7, A8, A9, A10, A11 and A12 genes in diverse
prostate tumor cell lines and prostate xenografts was analyzed by
RT-PCR. Shown are expression levels in individual samples tested.
Blank = no expression, + = low expression, ++ = high expression.
All cell lines/xenografts express at least one MAGE-A gene.
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S. Skov, M. H. Claesson. MHC-I-induced apoptosis in human
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Cao Y., Y. Lan, J. Qian, Y. Zheng, S. Hong, H. Li, M. Wang, L. W.
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.beta.2-microglobulin by pentameric IgM antibodies. Br. J.
Haematol. 2011, 154:111-121. [0129] McCurdy D. K., L. Q. Tai, K. L.
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2002, 29:2219-2224.
Sequence CWU 1
1
5212226DNAArtificialDNA sequence Hexa-AH5 1cagctgcagc tgcaagaaag
cggtggtggt gttgttcagc ctggtcgtag cctgcgtctg 60agctgtgcag caagcggttt
tacctttagc agctatggta tgcattgggt tcgtcaggca 120ccgggtaagg
aacgtgaagg tgttgcagtt attagctatg atggcagcaa caaatattat
180gccgatagcg ttaaaggtcg ctttaccatt agccgtgata atagcaaaaa
caccctgtat 240ctgcagatga atagcctgcg tgcagaagat accgcagttt
attattgtgc cggtggtagc 300tattatgttc cggattattg gggtcagggc
accctggtta ccgttagcag cggtagcacc 360agcggtagca tggcccagct
gcaattacaa gaatcaggtg gtggcgtggt gcagccaggt 420cgttcactgc
gtctgtcatg tgcagcatca ggctttacct tcagttcata cggcatgcac
480tgggtgcgcc aagctccagg caaagaacgc gaaggcgtgg ccgttatttc
atacgatggc 540tccaataaat actatgcgga ttcagtgaaa ggccgtttta
ccatttcacg cgataacagt 600aaaaacacct tatacctgca aatgaattca
ctgcgtgccg aggatacagc cgtgtattac 660tgtgcgggtg gttcatatta
cgtgcctgat tattggggac aaggtacact ggtgacagtt 720agcagtggta
gtacctcagg ttcaatggcc cagttacaac tgcaagaatc tggcggtggt
780gttgtgcaac cgggtcgctc tctgcgtctg agttgcgctg catcaggttt
tacattttca 840agctacggaa tgcactgggt tagacaggct cccggtaagg
aaagagaagg cgttgcggtt 900atcagttatg acggtagcaa taagtattat
gcggactctg ttaagggtcg ttttacaatt 960tctcgggaca atagcaagaa
tacactgtac ttacagatga actctctgag agcagaagat 1020acagccgtat
actattgcgc aggcggtagt tattatgtgc ctgactactg gggccaggga
1080acgctggtga ccgtgagtag cggttcaacc agcggttcaa tggcgcaact
gcaacttcaa 1140gagtctggtg gcggtgtggt acagcctggc cgttctctgc
gtttaagctg cgcagcctct 1200ggttttacgt tttcatctta tggaatgcat
tgggtacgcc aagcccctgg aaaagaacgt 1260gagggcgtag cagtgatctc
ttatgatggt tcgaacaaat attacgcgga ctccgtgaaa 1320ggacgcttta
caatctctcg tgataactca aaaaatacgc tgtatcttca aatgaactcc
1380ttacgtgcgg aagatactgc ggtctattac tgcgctggcg gttcttacta
tgtaccagat 1440tactggggac aggggacctt agttacagtt agctcaggta
gcaccagtgg ttctatggct 1500caattacagt tacaagaaag tggcggtggc
gtggtccaac ctggccgtag tctgcgcctg 1560tcttgcgcag cgagcggctt
tacattttct agttatggca tgcattgggt gagacaagct 1620ccggggaaag
agcgcgaagg ggttgcggtg atttcttatg acggcagtaa taaatactac
1680gcagatagtg tgaaaggtcg tttcacaatt agtcgcgata actccaaaaa
cacattatat 1740ttgcagatga acagtttgcg tgcggaggac acggctgtat
attattgtgc agggggttcc 1800tactatgtgc ccgactactg gggtcaaggg
accttagtga ccgtttcaag cggtagtacc 1860tctggtagta tggctcaact
tcagctgcaa gagtcaggcg gaggcgttgt ccagcctgga 1920cgctcactgc
gcttaagttg tgcagccagt ggctttacgt ttagctctta cgggatgcat
1980tgggtccggc aggcgcctgg gaaggaacgc gaaggtgtag ctgtgattag
ttacgatggc 2040agtaataagt attacgccga ttcagtaaaa ggtcgcttca
cgatttcgcg tgataattct 2100aagaataccc tttaccttca gatgaattcg
ttacgcgcag aggataccgc tgtatactac 2160tgtgctggcg gatcatatta
tgtcccagac tattgggggc agggtactct ggtaacggtt 2220agctct
22262123PRTArtificialAmino acid sequence AH5 2Gln Leu Gln Leu Gln
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Ser Tyr Tyr Val Pro Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Ser Thr
Ser Gly Ser 115 120 3121PRTArtificialAmino acid sequence apoptin
3Met Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe 1
5 10 15 Arg Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His Cys Arg
Glu 20 25 30 Ile Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser
Leu Cys Gly 35 40 45 Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser
Ala Thr Ala Asp Asn 50 55 60 Ser Glu Ser Thr Gly Phe Lys Asn Val
Pro Asp Leu Arg Thr Asp Gln 65 70 75 80 Pro Lys Pro Pro Ser Lys Lys
Arg Ser Cys Asp Pro Ser Glu Tyr Arg 85 90 95 Val Ser Glu Leu Lys
Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro 100 105 110 Arg Thr Ala
Lys Arg Arg Ile Arg Leu 115 120 45PRTArtificialLinker
peptideMISC_FEATURE(1)..(5)this sequence may be repeated n times,
where n is a positive integer 4Gly Gly Gly Gly Ser 1 5
56PRTArtificialLinker peptideMISC_FEATURE(1)..(6)this sequence may
be repeated n times, where n is a positive integer 5Gly Ser Thr Ser
Gly Ser 1 5 618PRTArtificialLinker peptide 6Gly Ser Thr Ser Gly Ser
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly
723PRTArtificialLinker peptide 7Glu Phe Ala Lys Thr Thr Ala Pro Ser
Val Tyr Pro Leu Ala Pro Val 1 5 10 15 Leu Glu Ser Ser Gly Ser Gly
20 810PRTArtificialIgG1 Linker peptide 8Glu Pro Lys Ser Cys Asp Lys
Thr His Thr 1 5 10 912PRTArtificialIgG3 Linker peptide 9Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr 1 5 10 107PRTArtificialIgG4
Linker peptide 10Glu Ser Lys Tyr Gly Pro Pro 1 5
119PRTArtificialAmino acid sequence MHC-1 HLA-A0201 presentable
peptide in MAGE-A 11Tyr Leu Glu Tyr Arg Gln Val Pro Gly 1 5
129PRTArtificialAmino acid sequence MCH-1 HLA-CW7 presentable
peptide in MAGE-A 12Glu Gly Asp Cys Ala Pro Glu Glu Lys 1 5
13121PRTArtificialAmino acid sequence Vh binding domain 11H 13Glu
Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Leu 35 40 45 Ser Tyr Ile Ser Ser Asp Gly Ser Thr Ile Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn
Ala Lys Asn Ser Leu Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Ser Pro Arg Gly
Tyr Tyr Tyr Tyr Gly Leu Asp Leu Trp Gly 100 105 110 Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 14119PRTArtificialAmino acid sequence
AH5 Vh binding domain 14Met Ala Gln Leu Gln Leu Gln Glu Ser Gly Gly
Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser Tyr Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ala Val Ile
Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Ala Gly Gly Ser Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 15117PRTArtificialAmino
acid sequence AH5 Vh binding domain 15Gln Leu Gln Leu Gln Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Gly Ser Tyr Tyr Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
1610PRTArtificialLinker peptide 16Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 1 5 10 17120PRTArtificialAmino acid sequence AH5 Vh binding
domain 17Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val
Phe Arg 1 5 10 15 Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His
Cys Arg Glu Ile 20 25 30 Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr
Leu Ser Leu Cys Gly Cys 35 40 45 Ala Asn Ala Arg Ala Pro Thr Leu
Arg Ser Ala Thr Ala Asp Asn Ser 50 55 60 Glu Ser Thr Gly Phe Lys
Asn Val Pro Asp Leu Arg Thr Asp Gln Pro 65 70 75 80 Lys Pro Pro Ser
Lys Lys Arg Ser Cys Asp Pro Ser Glu Tyr Arg Val 85 90 95 Ser Glu
Leu Lys Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro Arg 100 105 110
Thr Ala Lys Arg Arg Ile Arg Leu 115 120 1817PRTArtificialAmino acid
sequence cathepsin L cleavage site 18Arg Lys Glu Leu Val Thr Pro
Ala Arg Asp Phe Gly His Phe Gly Leu 1 5 10 15 Ser
19137PRTArtificialAmino acid sequence AH5 Vh binding domain 19Arg
Lys Glu Leu Val Thr Pro Ala Arg Asp Phe Gly His Phe Gly Leu 1 5 10
15 Ser Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe
20 25 30 Arg Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His Cys
Arg Glu 35 40 45 Ile Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu
Ser Leu Cys Gly 50 55 60 Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg
Ser Ala Thr Ala Asp Asn 65 70 75 80 Ser Glu Ser Thr Gly Phe Lys Asn
Val Pro Asp Leu Arg Thr Asp Gln 85 90 95 Pro Lys Pro Pro Ser Lys
Lys Arg Ser Cys Asp Pro Ser Glu Tyr Arg 100 105 110 Val Ser Glu Leu
Lys Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro 115 120 125 Arg Thr
Ala Lys Arg Arg Ile Arg Leu 130 135 2010PRTArtificialAmino acid
sequence cathepsin -B cleavage site 20Gly Phe Gln Gly Val Gln Phe
Ala Gly Phe 1 5 10 219PRTArtificialAmino acid sequence of MAGE-A3
peptide epitope binding to HLA 21Ile Met Pro Lys Ala Gly Leu Leu
Ile 1 5 229PRTArtificialAmino acid sequence MHC-1 HLA-A0201
presentable peptide in MAGE-A1 22Tyr Leu Glu Tyr Arg Gln Val Pro
Asp 1 5 239PRTArtificialAmino acid sequence MHC-1 HLA-A0201
presentable peptide in MAGE-A1, with enhanced binding capacity for
HLA-A0201 23Tyr Leu Glu Tyr Arg Gln Val Pro Val 1 5
2416PRTArtificialAmino acid sequence of MAGE-A3 peptide epitope
binding to HLA 24Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn
Tyr Leu Glu Tyr 1 5 10 15 259PRTArtificialAmino acid sequence of
MAGE peptide epitope binding to HLA 25Glu Ala Asp Pro Thr Gly His
Ser Tyr 1 5 269PRTArtificialamino acid sequence of MAGE peptide
epitope binding to HLA 26Ser Leu Phe Arg Ala Val Ile Thr Lys 1 5
279PRTArtificialamino acid sequence of MAGE peptide epitope binding
to HLA 27Asn Tyr Lys His Cys Phe Pro Glu Ile 1 5
2810PRTArtificialamino acid sequence of MAGE peptide epitope
binding to HLA 28Glu Val Tyr Asp Gly Arg Glu His Ser Ala 1 5 10
2910PRTArtificialamino acid sequence of MAGE peptide epitope
binding to HLA 29Arg Glu Pro Val Thr Lys Ala Glu Met Leu 1 5 10
309PRTArtificialamino acid sequence of MAGE peptide epitope binding
to HLA 30Asp Pro Ala Arg Tyr Glu Phe Leu Trp 1 5
319PRTArtificialamino acid sequence of MAGE peptide epitope binding
to HLA 31Ser Ala Phe Pro Thr Thr Ile Asn Phe 1 5
329PRTArtificialamino acid sequence of MAGE peptide epitope binding
to HLA 32Ser Ala Tyr Gly Glu Pro Arg Lys Leu 1 5
339PRTArtificialamino acid sequence of MAGE peptide binding to HLA
33Lys Met Val Glu Leu Val His Phe Leu 1 5 3410PRTArtificialamino
acid sequence of MAGE peptide binding to HLA 34Tyr Leu Gln Leu Val
Phe Gly Ile Glu Val 1 5 10 359PRTArtificialamino acid sequence of
MAGE peptide epitope binding to HLA 35Glu Tyr Leu Gln Leu Val Phe
Gly Ile 1 5 369PRTArtificialamino acid sequence of MAGE peptide
binding to HLA 36Glu Ala Asp Pro Ile Gly His Leu Tyr 1 5
379PRTArtificialamino acid sequence of MAGE peptide binding to HLA
37Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 3810PRTArtificialamino
acid sequence of MAGE peptide binding to HLA 38Met Glu Val Asp Pro
Ile Gly His Leu Tyr 1 5 10 399PRTArtificialamino acid sequence of
MAGE peptide binding to HLA 39Trp Gln Tyr Phe Phe Pro Val Ile Phe 1
5 4010PRTArtificialamino acid sequence of MAGE peptide binding to
HLA 40Gly Val Tyr Asp Gly Arg Glu His Thr Val 1 5 10
419PRTArtificialamino acid sequence of MAGE peptide binding to HLA
41Met Val Lys Ile Ser Gly Gly Pro Arg 1 5 429PRTArtificialamino
acid sequence of MAGE peptide binding to HLA 42Gly Leu Tyr Asp Gly
Met Glu His Leu 1 5 439PRTArtificialamino acid sequence of MAGE
peptide binding to HLA 43Val Arg Ile Gly His Leu Tyr Ile Leu 1 5
449PRTArtificialamino acid sequence of MAGE peptide binding to HLA
44Ala Ala Arg Ala Val Phe Leu Ala Leu 1 5 459PRTArtificialamino
acid sequence of DAM-6 and DAM-10 peptide epitope binding to HLA
45Phe Leu Trp Gly Pro Arg Ala Tyr Ala 1 5 468PRTArtificialamino
acid sequence of GAGE-1/-2/-8 peptide epitope binding to HLA 46Tyr
Arg Pro Arg Pro Arg Arg Tyr 1 5 479PRTArtificialamino acid sequence
of GAGE-3/-4/-5/-6/-7B peptide epitope binding to HLA 47Tyr Tyr Trp
Pro Arg Pro Arg Arg Tyr 1 5 4811PRTArtificialamino acid sequence of
NA88-A peptide epitope binding to HLA 48Met Thr Gln Gly Gln His Phe
Leu Gln Lys Val 1 5 10 4911PRTArtificialamino acid sequence of
NY-ESO-1 peptide epitope binding to HLA 49Ser Leu Leu Met Trp Ile
Thr Gln Cys Phe Leu 1 5 10 509PRTArtificialamino acid sequence of
NY-ESO-1a peptide epitope binding to HLA 50Ser Leu Leu Met Trp Ile
Thr Gln Cys 1 5 519PRTArtificialamino acid sequence of NY-ESO-1a
peptide epitope binding to HLA 51Gln Leu Ser Leu Leu Met Trp Ile
Thr 1 5 5210PRTArtificialamino acid sequence of NY-ESO-1a peptide
epitope binding to HLA 52Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg 1
5 10
* * * * *