NZ723544B2 - Claudin-6-specific immunoreceptors and t cell epitopes - Google Patents
Claudin-6-specific immunoreceptors and t cell epitopes Download PDFInfo
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- NZ723544B2 NZ723544B2 NZ723544A NZ72354415A NZ723544B2 NZ 723544 B2 NZ723544 B2 NZ 723544B2 NZ 723544 A NZ723544 A NZ 723544A NZ 72354415 A NZ72354415 A NZ 72354415A NZ 723544 B2 NZ723544 B2 NZ 723544B2
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The present invention provides Claudin-6-specific immunoreceptors (T cell receptors and artificial T cell receptors (chimeric antigen receptors; CARs)) and T cell epitopes which are useful for immunotherapy. In a particular embodiment, the T cell epitopes are peptides comprising an amino acid sequence selected from the group consisting of ALFGLLVYL (SEQ ID NO: 3), TLLGWVNGL (SEQ ID NO: 4) and QILGVVLTL (SEQ ID NO: 5) wherein the peptide is 100 or less amino acids long. ce selected from the group consisting of ALFGLLVYL (SEQ ID NO: 3), TLLGWVNGL (SEQ ID NO: 4) and QILGVVLTL (SEQ ID NO: 5) wherein the peptide is 100 or less amino acids long.
Description
CLAUDINSPECIFIC RECEPTORS AND T CELL EPITOPES
CAL FIELD OF THE lNVENTION
The present invention relates to the provision of Claudin-6—specific immunoreceptors (T cell
receptors and ial T cell receptors (chimeric antigen receptors; CARs)) and T cell es
which are useful for immunotherapy.
BACKGROUND OF THE INVENTION
The evolution of the immune system resulted in vertebrates in a highly effective network based
on two types of defense: the innate and the adoptive immunity.
In contrast to the evolutionary ancient innate immune system that relies on invariant receptors
recognizing common molecular patterns associated with pathogens, the adoptive immunity is
based on highly specific antigen receptors on B cells (B lymphocytes) and T cells (T
lymphocytes) and clonal selection.
While B cells raise humoral immune responses by secretion of antibodies, T cells mediate
cellular immune responses leading to destruction of recognized cells.
T cells play a central role in cell-mediated ty in humans and animals. The recognition
and g of a particular antigen is mediated by the T cell receptors (TCRs) expressed on the
surface of T cells.
The T cell receptor (TCR) of a T cell is able to ct with immunogenic peptides (epitopes)
bound to major histocompatibility complex (MHC) molecules and presented on the surface of
target cells. Specific binding of the TCR triggers a signal cascade inside the T cell leading to
proliferation and entiation into a maturated effector T cell. To be able to target a vast variety
of antigens, the T cell receptors need to have a great diversity.
This diversity is ed by genetic rearrangement of different discontinuous segments of
genes
which code for the ent structural regions of TCRs. TCRs are composed of one u—chain and
one B-chain or of one y—chain and one 8-chain. The TCR a/B chains are composed of an N-
terminal highly polymorphic variable region involved in antigen recognition and an invariant
constant region. On the genetic level, these chains are ted into several regions, a variable
(V) region, a diversity (D) region (only [3- and 8-chain), a joining (J) region and a constant (C)
region. The human n genes n over 60 variable (V), 2 diversity (D), over 10 g
(J) segments, and 2 constant region segments (C). The human a-chain genes contain over 50 V
segments, and over 60 J segments but no D segments, as well as one C segment. The murine [3-
chain genes contain over 30 variable (V), 2 diversity (D), over 10 g (J) segments, and 2
constant region segments (C). The murine a-chain genes contain almost 100 V segments, 60 J
segments, no D segments, but one C segment. During the differentiation of T cells, specific T
cell receptor genes are created by nging one V, one D (only [3— and S-chain),
one J and one
C region gene. The diversity of the TCRs is further amplified by imprecise V-(D)-J
ieerrangement wherein random nucleotides are introduced and/or deleted at the ination
sites. Since the rearrangement of the TCR gene loci occurs in the
genome during maturation of T
cells, each mature T cell only expresses one specific (it/B TCR or 7/5 TCR.
MHC and antigen binding is mediated by the complementary determining regions 1, 2 and 3
(CDRl, CDRZ, CDR3) of the TCR. The CDR3 of the B-chain which is most critical for antigen
recognition and binding is encoded by the V-D-J junction of the rearranged TCR B—chain gene.
The TCR is a part of a complex signaling ery, which includes the heterodimeric complex
of the TCR a,- and B-chains, the eptor CD4 or CD8 and the CD3 signal transduction modul
(Figure 1). While the CD3 chains transfer the activation signal inside the cell, the TCR (l/B
heterodimer is solely responsible for antigen recognition. Thus, the transfer of the TCR a/B
chains offers the opportunity to ct T cells towards
any antigen of interest.
Immunotherapy
Antigen-specific immunotherapy aims to enhance or induce specific immune responses in
ts to control infectious or malignant diseases. The identification of a growing number of
en- and tumor—associated antigens (TAA) led to a broad collection of suitable targets for
therapy. Cells presenting immunogenic peptides (epitopes) derived from these antigens
can be specifically ed by either active or passive zation strategies.
Active immunization tends to induce and expand antigen-specific T cells in the patient, which
are able to specifically recognize and kill diseased cells. In contrast passive immunization relies
on the adoptive transfer of T cells, which were expanded and optional genetically engineered in
vitro (adoptive T cell therapy).
Vaccination
Tumor vaccines aim to induce endogenous tumor-specific immune ses by active
immunization. Different n formats can be used for tumor vaccination including whole
cancer cells, ns, es or immunizing vectors such as RNA, DNA or viral vectors that
can be applied either directly in vivo or in vitro by pulsing of DCs following transfer into the
patient.
The number of clinical s where therapy-induced immune responses can be identified is
steadily increasing due to ements of immunization strategies and methods for detection of
antigen—specific immune ses (Connerotte, T. et a1. (2008). Cancer Res. 68, 3931-3940;
Schmitt, M. et a1. (2008) Blood 111, 1357-1365; Speiser, D.E. et al. (2008) Proc. Natl. Acad.
Sci. U. S. A 105, 3849-3854; Adams, S. et al. (2008) J. Immunol. 181, 776-784).
However, in most cases detected immune responses cannot systemically be correlated with
clinical outcomes (Curigliano, G. et al. (2006) Ann. Oncol. 17, 750—762; Rosenberg, S.A. et al.
(2004) Nat. Med. 10, 909—915).
The exact definition of peptide epitopes derived from tumor antigens
may therefore contribute to
e specificity and efficiency of vaccination strategies as well as methods for
immunornonitoring.
Adoptive cell transfer (ACT)
ACT based immunotherapy can be broadly defined as a form of passive immunization with
previously sensitized T cells that are transferred to non—immune recipients or to the autologous
host after ex Vivo expansion from low precursor ncies to clinically relevant cell numbers.
Cell types that have been used for ACT experiments are lymphokine—activated killer (LAK) cells
(Mule, J.J. et al. (1984) Science 225, 1487-1489; Rosenberg, S.A. et al. (1985) N. Engl. J. Med.
313, 1485—1492), tumor-infiltrating lymphocytes (TILs) (Rosenberg, S.A. et al. (1994) J. Natl.
Cancer Inst. 86, 166), donor lymphocytes after hematopoietic stem cell transplantation
(HSCT) as well as tumor-specific T cell lines or clones (Dudley, M.E. et al. (2001) J.
Immunother. 24, 3; Yee, C. et a1. (2002) Proc. Natl. Acad. Sci. U. S. A 99, 16168-16173).
Adoptive T cell transfer was shown to have therapeutic activity against human viral infections
such as CMV. While CMV infection and reactivation of endogenous latent viruses is controlled
by the immune system in healthy individuals, it results in significant ity and mortality in
immune compromised individuals such as transplant recipients or AIDS patients.
2015/056899
Riddell and co-workers demonstrated the reconstitution of viral immunity by ve T cell
therapy in immune suppressed ts after transfer of CD8+ ecific T cell clones
derived from HLA—matched CMV—seropositive transplant donors (Riddell, SR. (1992) Science
257, 238-241).
As an alternative approach onal donor—derived CMV- or EBV—specific T cell populations
were transferred to transplant recipients resulting in increased persistence of transferred T cells
(itooney, CM. et al. (1998) Blood 92, 154‘7-1555; Peggs, KS. "t al. (2003) Lancet 362, 1375-
1377).
For adoptive immunotherapy of melanoma Rosenberg and co-workers established an ACT
approach relying on the infusion of in Vitro expanded autologous tumor-infiltrating lymphocytes
(TILs) ed from excised tumors in combination with a non—myeloablative lymphodepleting
chemotherapy and high-dose 1L2. A recently published clinical study resulted in an objective
response rate of ~50% of treated patients suffering from metastatic ma (Dudley, M.E. et
a1. (2005) J. Clin. Oncol. 23: 2346-2357).
However, patients must fulfill several premises to be eligible for ACT therapy. They
must have resectable tumors. The tumors must generate viable TILs under cell culture
conditions. The TILS must be reactive against tumor antigens, and must expand in vitro to
sufficient numbers. Especially in other cancers than melanoma, it is difficult to obtain such
tumor—reactive TILs. Furthermore, repeated in vitro stimulation and clonal expansion of normal
human T lymphocytes results in progressive decrease in telomerase activity and shortening of
telomeres resulting in replicative senescence and decreased ial for persistence of
erred T cells (Sheri, X. et al. (2007) J. Immunother. 30: 123-129).
ACT using ngineered T cells
An approach ming the limitations of ACT is the adoptive transfer of autologous T cells
reprogrammed to express a tumor-reactive immunoreceptor of defined specificity during short-
time ex vivo culture followed by sion into the patient (Kershaw M.H. et a1. (2013) Nature
Reviews Cancer 13 5-41). This strategy makes ACT applicable to a variety of common
malignancies even if tumor-reactive T cells are absent in the patient. Since the nic
specificity of T cells is rested entirely on the heterodimeric complex of the TCR 0t— and B—chain,
the transfer of cloned TCR genes into T cells offers the potential to redirect them towards
antigen of interest. Therefore, TCR gene therapy provides an attractive gy to develop
antigen-specific immunotherapy with autologous lymphocytes as treatment option. Major
advantages of TCR gene transfer are the creation of therapeutic quantities of antigen-specific T
cells within a few days and the possibility to introduce specificities that are not present in the
endogenous TCR repertoire of the patient.
Several groups demonstrated, that TCR gene transfer is an attractive strategy to redirect antigen-
specificity of primary T cells (Morgan, RA et a1. (2003) J. Immunol. 171, 3287-3295; Cooper,
L]. et al. (2000) J. Virol. 74, 8207-8212; Fujio, K. et a1. (2000) J. Immunol. 165, 528-532;
Kessels, HWY et a1. (2001) Nat. Immunol. 2, 957~961; Dembic, Z. et al. (1986) Nature 320, 232-
238)
Feasibility of TCR gene y in humans was recently demonstrated in clinical trials for the
treatment of malignant melanoma by Rosenberg and his group. The adoptive transfer of
autologous lymphocytes retrovirally transduced with melanoma/melanocyte antigen-specific
TCRs resulted in cancer regression in up to 30% of treated melanoma patients (Morgan, RA. et
al. (2006) e 314, 126-129; Johnson, L.A. et a1. (2009) Blood 1 14, 535-546).
Chimeric n ors
Chimeric antigen receptors (CARS) are engineered receptors that combine a single chain variable
fragment (scFV) of a monoclonal antibody with an intracellular part consisting of one or more
signaling domains for T cell activation. CARS recognize native antigens in a non—MHC-restricted
manner and can therefore be used in all individuals no matter what their HLA type is and they
are functional in CD4+ as well as CD8+ T cells.
A multitude of CARS has been reported over the past decade, targeting a panel of different cell
surface tumor antigens. Their biologic functions were dramatically improved by incorporation of
a costimulatory domain resulting in tite receptors (scFv, CD28, CD30, termed 2nd
tion CARS. CARS of the 3rd generation ass additional s of costimulatory
molecules such as 0X40 and 4-1BB to enhance the proliferative capacity and persistence of
modified T—cells (Figure 2).
Target ures for antigen-specific therapy
The ery of multiple tumor-associated antigens (TAAS) has provided the basis for antigen-
specific immunotherapy concepts lino, L. et a1. (2005) Cancer Immunol. Immunother. 54,
187-207). TAAs are unusual ns expressed on tumor cells due to their genetic instability,
which have no or limited expression in normal cells. These TAAs can lead to specific
recognition of malignant cells by the immune system.
Molecular cloning of TAAS by screening of tumor-derived cDNA expression libraries using
autologous tumor-specific T cells (van der Bruggen, P. et al. (1991) Science 254, 1643-1647) or
circulating antibodies (Sahin, U. et a1. (1995) Proc. Natl. Acad. Sci. U. S. A 92, 11810—11813),
e immunology approaches, biochemical methods (Hunt, D.F. et a1. (1992) Science 256,
820), gene expression analyses or in silico cloning gies enbein, G. et a1. (2008)
Gene 414, 76-84) led to a significant number of target candidates for immunotherapeutic
strategies. TAAs fall in several categories, including differentiation antigens, pressed
antigens, tumor-specific splice variants, mutated gene products, viral and cancer testis antigens
(CTAs). The cancer testis family is a very promising category of TAAs as their expression is
restricted to the testis and a multitude of different tumor entities (Scanlan, M.J. et a1. (2002)
Immunol. Rev. 188, 22-32). Until now more than 50 CT genes have been described (Scanlan,
M.J. et a1. (2004) Cancer Immun. 4, l) and some of them have been addressed in clinical s
(Adams, S. et a1. (2008) J. Immunol. 181, 776-784; Atanackovic, D. et a1. (2004) J. Immunol.
172, 3289—3296; Chen, Q. et a1. (2004) Proc. Natl. Acad. Sci. U. S. A 101, 9363-9368;
otte, T. et al. (2008). Cancer Res. 68, 3931—3940; Davis, LD. et al. (2004) Proc. Natl.
Acad. Sci. U. S. A 101, 10697-10702; Jager, E. (2000) Proc. Natl. Acad. Sci. U. S. A 97, 12198—
12203; Marchand, M. et al. (1999) Int. J. Cancer 80, 219-230; Schuler-Thurner, B. et al. (2000)
J. Immunol. 165, 496).
In spite of the growing number of attractive target structures for immunotherapeutic approaches
specific T cell clones or lines of defined HLA restriction do only exist for a few of them (Chaux,
P. et al. (1999) J. Immunol. 163, 2928—2936; Zhang, Y. et a1. (2002) Tissue Antigens 60, 365-
371; Zhao, Y. et a1. (2005) J. Immunol. 174, 4415—4423).
ns are integral membrane proteins located within the tight junctions of epithelia and
endothelia. Claudins are predicted to have four transmembrane segments with two extracellular
loops, and N- and C-termini d in the asm. The Claudin (CLDN) family of
transmembrane proteins plays a critical role in the maintenance of epithelial and endothelial tight
junctions and might also play a role in the maintenance of the cytoskeleton and in cell signalling.
2015/056899
Claudin-6 (CLDN6) is an oncofetal gene expressed in murine and human stem cells as well as
embryoid bodies committed to the epithelia cell fate (Turksen, K. et a1. (2001) Dev Dyn 222,
292-300; Anderson WJ. et a1. (2008) Dev Dyn 237, ; Turksen K. et al. (2002)
Development, 129, 4; Assou S. et a1. (2007) Stem Cells 25, 961—73). As a tumor-
associated antigen it can be classified as a differentiation antigen due to its expression during
early stage of epidermal morphogenesis where it is crucial for epidermal entiation and
barrier formation. Additionally expression was observed in epithelial tissues or neonatal normal
epithelial tissue of tongue, skin, stomach and breast (Abuazza G. et al. (2006), Am J l
Renal Physiol 291, 1132-1141; Troy T.C. et a1. (2007), Molecular Biotechnology 36, 166—74;
Zhao L. ct a1. (2008), Am J Physiol Regul Integr Comp Physiol 294, 1856-1862). Besides that,
own data also reveal low or very low expression of CLDN6 in human placenta, urinary bladder,
endometrium, prostate and the peripheral nerve and frequent overexpression of CLDN6 in
different cancers. CLDN6 has been demonstrated to be overexpressed in tumors, including
pediatric brain tumors, gastric adenocarcinomas and germ cell tumors as well as visceral
carcinomas such as ovarian omas (Figure4). It has also been demonstrated that
overexpression of CLDN6 in gastric cancer cells s in increased invasiveness, migration and
proliferation suggesting that CLDN6 is a marker for poor prognosis and may play a potential role
in ining the malignant phenotype. In addition, it has been shown that CLDN6 functions as
cancer suppressor via inhibition of cell eration and induction of apoptosis in breast cancer
cell lines.
The frequent overexpression of CLDN6 on tumors qualifies this molecule as a highly attractive
target for development of therapeutics directed against CLDN6 such as vaccine therapeutics and
therapeutic antibodies. However, hitherto no 2-restricted CLDN6 T cell epitopes and T
cell receptors ing CLDN6 have been described and it is unknown whether CLDN6
expressing cancer cells can be ed in vivo by immunotherapies involving T cells using
active or passive immunization approaches.
DESCRIPTION OF INVENTION
Summary of the invention
The present invention s to T cell receptors and artificial T cell receptors specific for the
tumor-associated antigen CLDN6, in particular when present on the surface of a cell such as
diseased cell or presented on the surface of a cell such as a diseased cell or an n-presenting
cell, as well as peptides sing es recognized by these T cell receptors, i.e. CLDN6—T
cell epitopes.
By adoptive transfer of T cells engineered to s such T cell or or ial T cell
receptor CLDN6 expressing cancer cells can be specifically targeted thereby leading to selective
destruction of cancer cells. Furthermore, the T cell epitopes provided according to the invention
are useful for designing vaccines against CLDN6-expressing cancers.
In one aspect, the invention relates to a peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS: 3, 4 and 5 or a variant of said amino acid
sequence.
In one embodiment the e is 100 or less, 50 or less, 20 or less, or 10 or less amino acids
long. In one embodiment, the peptide can be processed to produce a peptide consisting of the
amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4 and 5 or a variant
of said amino acid sequence. In one embodiment, the peptide consists of the amino acid
sequence selected from the group consisting of SEQ ID NOs: 3, 4 and 5 or a variant of said
amino acid sequence.
In one embodiment, the peptide is a MHC class I or class 11 presented peptide, preferably a MHC
class I presented peptide, or, if present within cells, can be processed to produce a sion
product thereof which is a MHC class I or class 11 presented peptide, ably a MHC class I
ted peptide. Preferably, said MHC class I or class II ted peptide has a sequence
substantially corresponding to the given amino acid ce, i.e. an amino acid sequence
selected from the group consisting of SEQ ID NOS: 3, 4 and 5 or a variant of said amino acid
sequence. Preferably, a peptide according to the invention is capable of stimulating a cellular
response against a disease involving cells characterized by presentation of CLDN6 with class I
MHC.
In further aspects, the invention relates to a nucleic acid comprising a nucleotide sequence
encoding the peptide of the invention and a cell comprising the nucleic acid. The nucleic acid
may be a recombinant nucleic acid. The nucleic acid may be present in a plasmid or an
expression vector and may be functionally linked to a promoter. In one embodiment, the nucleic
acid is RNA. Preferably, the cell expresses the peptide. The cell may be a recombinant cell and
may e the encoded peptide or a procession product thereof, may express it on the surface
and preferably may additionally express an MHC molecule which binds to said peptide or a
procession product thereof and preferably presents said peptide or a procession product f
on the cell surface. In one embodiment, the cell ses the MHC molecule endogenously. In a
firrther embodiment, the cell expresses the MHC molecule and/or the peptide in a recombinant
manner. The cell is preferably nonproliferative. In a preferred embodiment, the cell is an antigen-
presenting cell, in particular a dendritic cell, a monocyte or a macrophage.
In a further aspect, the invention s to a cell that presents the e of the ion or a
procession product thereof, wherein the procession product preferably is a e having the
given amino acid sequence, i.e. an amino acid ce selected from the group consisting of
SEQ ID NOS: 3, 4 and 5 or a variant of said amino acid sequence. In one embodiment, said cell
is a cell comprising a nucleic acid comprising a nucleotide sequence encoding the peptide of the
invention. Preferably said cell expresses said c acid so as to produce said peptide.
Optionally said cell processes said peptide so as to produce a peptide consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 3, 4 and S or a variant of said
amino acid sequence. The cell may present the peptide or a procession product thereof by MHC
molecules on its surface. In one embodiment, the cell endogenously expresses an MHC
le. In a further embodiment, the cell recombinantly expresses an MHC molecule. In one
embodiment, the MHC molecules of the cell are loaded (pulsed) with the peptide by addition of
the e to the cell. The cell may recombinantly express the peptide and present said peptide
or a procession product thereof on the cell e. The cell is preferably nonproliferative. In a
preferred embodiment, the cell is an antigen-presenting cell such as a tic cell, a monocyte
or a macrophage.
In a further aspect, the invention relates to an immunoreactive cell which is reactive with a
peptide of the invention, in particular when presented on the surface of a cell such as a diseased
cell. The reactive cell may be a cell that has been sensitized in vitro to recognize the
peptide. The immunoreactive cell may be a T cell, preferably a cytotoxic T cell. Preferably, the
immunoreactive cell binds to a sequence substantially corresponding to the given amino acid
sequence, ie. an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4
and 5 or a variant of said amino acid sequence, in particular when bound to MHC such as MHC
on the surface of a cell such as a diseased cell.
In a further aspect, the invention relates to a binding agent which binds to a peptide of the
invention, ally in a complex with an MHC molecule.
In a further aspect, the invention relates to a T cell receptor which binds to a peptide of the
invention, optionally in a complex with an MHC molecule, and preferably is reactive with said
peptide, or a polypeptide chain of said T cell receptor. In one embodiment, the polypeptide chain
of said T cell or is a T cell receptor a-chain or T cell receptor n.
In a further aspect, the invention relates to a T cell receptor (it—chain or a T cell receptor
comprising said T cell receptor (ii-chain,
wherein said T cell receptor a—chain is selected from the group consisting of:
(i) a T cell receptor (it-chain comprising at least one, preferably two, more preferably all three of
the CDR sequences of a T cell receptor Q-Chaln selected from the group consisting of SEQ ID
NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 or a variant thereof and
(ii) a T cell receptor tit—chain comprising a T cell receptor tit-chain sequence selected from the
group consisting of SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 or a fragment
thereof, or a variant of said sequence or fragment.
In one ment, said SEQ ID NOs: are selected from the group consisting of SEQ ID NOS:
6, 8, 10, 12, 14 and 16 and said T cell receptor is reactive with a peptide comprising the amino
acid ce of SEQ ID NO: 3 or a variant of said amino acid sequence.
In one embodiment, said SEQ ID NOs: are selected from the group consisting of SEQ ID N05:
18, 20, 22, 24 and 26 and said T cell receptor is reactive with a peptide comprising the amino
acid ce of SEQ ID NO: 4 or a t of said amino acid ce.
In one embodiment, said SEQ ID NO: is SEQ ID NO: 28 and said T cell receptor is reactive with
a e comprising the amino acid ce of SEQ ID NO: 5 or a variant of said amino acid
sequence.
In a further aspect, the invention relates to a T cell receptor B-chain or a T cell receptor
sing said T cell receptor B—chain,
wherein said T cell receptor B-chain is selected from the group consisting of:
(i) a T cell receptor B-chain comprising at least one, preferably two, more preferably all three of
the CDR sequences of a T cell receptor B-chain ed from the group consisting of SEQ ID
NOs:7,9,11, 13,15, 17,19, 21, 23, 25, 27 and 29 or a variant thereof
(ii) a T cell receptor B-chain comprising a T cell receptor B-chain sequence selected from the
group consisting of SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29 or a fragment
thereof, or a variant of said sequence or fragment.
In one embodiment, said SEQ ID NOS: are selected from the group consisting of SEQ ID NOS:
7, 9, 11, 13, 15 and 17 and said T cell receptor is reactive with a peptide comprising the amino
acid sequence of SEQ ID NO: 3 or a variant of said amino acid sequence.
In one embodiment, said SEQ ID NOS: are selected from the group consisting of SEQ ID NOS:
19, 21, 23, 25 and 27 and said T cell receptor is ve with a peptide sing the amino
acid sequence of SEQ ID NO: 4 or a variant of said amino acid sequence.
In one embodiment, said SEQ ID NO: is SEQ ID NO: 29 and said T cell or is reactive with
a e sing the amino acid sequence of SEQ ID NO: 5 or a variant of said amino acid
SCqUCDCC.
In a further aspect, the invention relates to a T cell receptor selected from the group consisting
(I) a T cell receptor comprising:
(i) a T cell receptor (it-chain comprising at least one, preferably two, more preferably all three of
the CDR ces of the T cell receptor (it-chain of SEQ ID NO: x or a variant thereof, and
(ii) a T cell receptor B-chain comprising at least one, ably two, more preferably all three of
the CDR ces of a T cell receptor B-chain of SEQ ID NO: x+l or a variant thereof;
wherein X selected from the group consisting of 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28
(II) a T cell or sing:
(i) a T cell receptor (It—chain comprising the T cell receptor (it-chain sequence of SEQ ID NO: x or
a fragment thereof, or a variant of said sequence or fragment, and
(ii) a T cell receptor B-chain comprising the T cell receptor B-chain sequence of SEQ ID NO:
x+1 or a fragment thereof, or a variant of said sequence or fragment;
wherein x selected from the group consisting of 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.
In one embodiment, said x is selected from the group consisting of 6, 8, 10, 12, 14 and 16 and
said T cell receptor is reactive with a peptide comprising the amino acid
sequence of SEQ ID
NO: 3 or a variant of said amino acid ce.
In one embodiment, said x is ed from the group consisting of l 8, 20, 22, 24 and 26 and said
T cell receptor is reactive with a peptide comprising the amino acid
ce of SEQ ID NO: 4
or a variant of said amino acid sequence.
In one embodiment, said x is 28 and said T cell receptor is reactive with a e comprising the
amino acid sequence of SEQ ID NO: 5 or a variant of said amino acid
sequence.
In one embodiment, binding of said T cell receptor when expressed by T cells and/or present on
T cells to peptide epitopes as described above presented on cells such as cancer cells
results in proliferation and/or activation of said T cells, wherein said activated T cells preferably
release cytotoxic factors, e. g. perforins and granzymes, and initiate cytolysis and/or apoptosis of
cancer cells.
In a further aspect, the invention relates to an artificial T cell or which binds to claudin-6
(CLDN6). In one embodiment, binding is a specific binding.
In one embodiment, said CLDN6 is expressed in a cancer cell. In one embodiment said CLDN6
is expressed on the surface of a cancer cell. In one embodiment said ial T cell receptor
binds to an extracellular domain or to an epitope in an extracellular domain of CLDN6. In one
embodiment said ial T cell receptor binds to native epitopes of CLDN6 present on the
surface of living cells. In one embodiment said artificial T cell receptor binds to the first
extracellular loop of CLDN6. In one embodiment, binding of said artificial T cell receptor when
expressed. by T cells and/or present on T cells to CLDN6 present on cells such as cancer cells
results in proliferation and/or activation of said T cells, wherein said activated T cells preferably
release cytotoxic s, e. g. ins and granzymes, and initiate cytolysis and/or apoptosis of
cancer cells.
WO 50327 2015/056899
In one embodiment, the artificial T cell receptor of the invention comprises a binding domain for
CLDN6. In one embodiment, the g domain for CLDN6 is comprised by an exodornain of
said artificial T cell receptor. In one embodiment, the binding domain for CLDN6 comprises a
single—chain variable fragment (scFv) of a CLDN6 antibody. In one embodiment, the binding
domain for CLDN6 comprises a variable region of a heavy chain of an immunoglobulin (VH)
with a specificity for CLDN6 (VH(CLDN6)) and a variable region of a light chain of an
immunoglobulin (VL) with a specificity for CLDN6 (VL(CLDN6)). In one embodiment, said
heavy chain variable region (VH) and the corresponding light chain variable region (VL) are
connected via a peptide , ably a peptide linker comprising the amino acid sequence
(GGGGS)3. In one embodiment, the binding domain for CLDN6 comprises a VH(CLDN6)
comprising an amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, or a
variant of said amino acid sequence or fragment. In one embodiment, the binding domain for
CLDN6 comprises a VL(CLDN6) comprising an amino acid ce represented by SEQ ID
NO: 33, 38 or 39 or a fragment thereof, or a variant of said amino acid sequence or fragment. In
one embodiment, the binding domain for CLDN6 comprises a VH(CLDN6) comprising an
amino acid sequence represented by SEQ ID NO: 32 or a fragment thereof, or a variant of said
amino acid sequence or fragment and a VL(CLDN6) comprising an amino acid sequence
represented by SEQ ID NO: 39 or a fragment thereof, or a variant of said amino acid sequence or
fragment. In one embodiment, the binding domain for CLDN6 comprises an amino acid
sequence represented by SEQ ID NO: 40 or a fragment thereof, or a variant of said amino acid
sequence or fragment.
In one embodiment, the artificial T cell receptor of the invention comprises a transmembrane
domain. In one embodiment, the transmembrane domain is a hobic alpha helix that spans
the membrane. In one ment, the transmembrane domain comprises the CD28
embrane domain or a nt thereof.
In one embodiment, the artificial T cell receptor of the invention comprises a T cell ing
. In one embodiment, the T cell signaling domain is located intracellularly. In one
embodiment, the T cell signaling domain comprises CD3-zeta, preferably the endodomain of
CD3-Zeta, optionally in combination with CD28. In one embodiment, the T cell ing
domain comprises the sequence according to SEQ ID NO: 45 or a fragment thereof, or a t
of said sequence or fragment.
In one embodiment, the artificial T cell receptor of the ion comprises a signal peptide
which directs the nascent protein into the endoplasmic reticulum. In one embodiment, the signal
e precedes the binding domain for CLDN6. In one embodiment, the signal peptide
comprises the sequence according to SEQ ID NO: 42 or a fragment thereof, or a variant of said
sequence or nt.
In one embodiment, the artificial T cell receptor of the ion comprises a spacer region
which links the binding domain for CLDN6 to the transmembrane domain. In one ment,
the spacer region allows the binding domain for CLDN6 to orient in different directions to
facilitate CLDN6 recognition. In one embodiment, the spacer region comprises the hinge region
from IgGl. In one embodiment, the spacer region comprises the sequence according to SEQ ID
NO: 43 or a fragment thereof, or a variant of said ce or nt.
In one embodiment, the ial T cell receptor of the invention comprises the structure:
NH2 — signal peptide - binding domain for CLDN6 - spacer region - transmembrane domain - T
cell signaling domain — COOH.
In one embodiment, the artificial T cell receptor of the invention comprises the amino acid
sequence according to SEQ ID NO: 46 or a fragment thereof, or a t of said amino acid
sequence or fragment.
The above T cell receptors and artificial T cell receptors are preferably specific for the tumor=
associated antigen CLDN6, in particular when present on the surface of a cell such as a diseased
cell or when presented on the surface of a cell such as a diseased cell or an antigen-presenting
cell.
The T cell receptors and artificial T cell receptors of the invention may be expressed by and/or
present on the surface of cells such as T cells.
In a further aspect, the invention relates to a nucleic acid comprising a nucleotide ce
encoding the T cell receptor chain or T cell receptor of the invention or encoding the artificial T
cell receptor of the invention. In one ment, the nucleic acid is a recombinant nucleic acid.
In one embodiment, the nucleic acid is in the form of a vector or in the form of RNA.
In a further aspect, the invention relates to a cell comprising the T cell receptor chain or T cell
receptor of the invention or the artificial T cell receptor of the invention and/or comprising a
nucleic acid comprising a nucleotide ce ng the T cell receptor chain or T cell
receptor of the invention or encoding the artificial T cell receptor of the invention. In one
embodiment, said nucleic acid is RNA, preferably in vitro transcribed RNA. The cell may be a
cell sing the T cell or chain or T cell receptor of the invention or the artificial T cell
or of the invention and/or may have the T cell receptor chain or T cell receptor of the
invention or the artificial T cell receptor of the invention on its cell surface. In one embodiment,
said cell is a cell which is useful for adoptive cell er. The cell may be an effector or stem
cell, preferably an immunoreactive cell. The reactive cell may be a T cell, preferably a
cytotoxic T cell. In one ment, the immunoreactive cell is reactive with the tumor—
associated antigen CLDN6. In one embodiment, said CLDN6 is present on the surface of a cell
such as a diseased cell. In one embodiment, said CLDN6 is presented on the surface of a cell
such as a diseased cell or an antigen-presenting cell, and the immunoreactive cell is reactive with
a peptide of the invention, in particular when presented in the context of MHC, and preferably
binds to a sequence ntially corresponding to the given amino acid sequence, i.e. an amino
acid sequence selected from the group consisting of SEQ ID NOS: 3, 4 and 5 or a variant of said
amino acid sequence. In one embodiment, said cell lacks surface expression of an endogenous
TCR or is specific for a CLDN6—unrelated n.
In one embodiment, cells of the invention prior to use in adoptive cell transfer are ted to an
antigen-specific expansion and rechallenge, wherein the antigen-specific expansion and
rechallenge may be effected by exposing the cells to ably autologous antigen presenting
cells presenting CLDN6 or a peptide fragment thereof.
In a further aspect, the invention relates to a method of producing an immunoreactive cell
comprising the step of transducing a T cell with a nucleic acid comprising a nucleotide sequence
encoding the T cell receptor chain or T cell receptor of the invention or encoding the artificial T
cell receptor of the ion.
Furthermore, the present invention generally embraces the treatment of diseases by targeting
diseased cells such as cancer cells, in particular cancer cells expressing CLDN6. The methods
provide for the ive eradication of cells that express on their surface and/or present the
2015/056899
tumor-associated n CLDN6, thereby minimizing adverse effects to normal cells not
sing and/0r presenting CLDN6. Thus, preferred diseases for a therapy are those in which
CLDN6 is expressed and optionally presented such as cancer diseases, in particular those
described herein.
When a peptide of the invention, a nucleic acid comprising a tide sequence encoding the
peptide of the invention or a cell of the invention comprising said nucleic acid is administered,
the treatment preferably involves an active immunization. Preferably, CLDN6-specific T cells
are expanded in the patient, which are able to ize and kill diseased cells. When an
immunoreactive cell of the invention, a T cell receptor of the invention, an artificial T cell
receptor of the invention, a nucleic acid of the invention comprising a nucleotide sequence
encoding a T cell receptor of the invention or encoding an artificial T cell or of the
invention or a cell of the invention comprising a T cell receptor or an artificial T cell receptor of
the invention and/0r comprising a nucleic acid of the invention comprising a nucleotide sequence
ng a T cell receptor of the invention or ng an artificial T cell receptor of the
invention is administered, the treatment preferably involves a passive immunization. Preferably,
specific T cells which are able to ize and kill diseased cells and which were
optionally genetically ered and/or expanded in vitro are adoptively transferred to a patient.
In one , the invention relates to a pharmaceutical ition comprising one or more of:
(i) the peptide of the invention;
(ii) the nucleic acid of the invention;
(iii) the cell of the invention;
(iv) the immunoreactive cell of the invention;
(v) the binding agent of the invention;
(vi) the T cell receptor of the invention; and
(vi) the artificial T cell receptor of the invention.
A pharmaceutical composition of the invention may comprise a pharmaceutically acceptable
carrier and may ally comprise one or more adjuvants, stabilizers etc. The pharmaceutical
composition may in the form of a therapeutic or prophylactic vaccine. In one embodiment, the
pharmaceutical composition is for use in treating or preventing a cancer disease such as those
described herein.
Administration of a pharmaceutical composition as bed above
may provide MHC class 11—
presented epitopes that are capable of eliciting a CD4+ helper T cell response and/or a CD8+ T
cell response against CLDN6 (including cells expressing CLDN6 on their surface and/or
presenting CLDN6 in the context of MHC les). Alternatively or additionally,
administration of a pharmaceutical composition as described above may provide MHC class I-
presented epitopes that are capable of ing a CD8+ T cell se against CLDN6.
in a further aspect, the invention relates to a method of treating or preventing a cancer disease
comprising administering to a patient the ceutical composition of the invention.
In a further aspect, the invention relates to the peptide of the invention, the nucleic acid of the
invention, the cell of the invention, the immunoreactive cell of the invention, the binding agent
of the invention, the T cell receptor of the invention, or the ial T cell receptor of the
invention for use in therapy, in particular for use in treating or preventing cancer.
Another aspect relates to a method for inducing an immune response in a subject, comprising
administering to the subject a pharmaceutical composition of the invention.
Another aspect relates to a method for stimulating, priming and/or ing T cells, comprising
contacting T cells with one or more of: the peptide of the invention, the nucleic acid of the
invention comprising a nucleotide ce encoding the e of the invention, the cell of the
ion comprising said nucleic acid and/or the cell of the invention that presents the peptide
of the invention or a procession product thereof. In one embodiment, the peptide of the ion
is presented in the context of MHC molecules such as MHC molecules on the surface of cells,
e.g. antigen-presenting cells.
In this aspect, the invention may relate to a method for preparing CLDN6-specific T cells. The T
cells may be stimulated, primed and/or expanded in vitro or in vivo. Preferably, the T cells are
present in a sample obtained from a subject. The stimulated, primed and/0r expanded T cells may
be administered to a subject and may be autologous, allogeneic, syngeneic to the subject.
The invention in the above aspects of a method for inducing an immune
response in a subject or
of a method for stimulating, priming and/or expanding T cells may relate to a method for treating
cancer diseases in a subject.
Another aspect relates to a method of killing cancer cells in a t, comprising the step of
providing to the subject a therapeutically effective amount of the peptide of the invention, the
nucleic acid of the invention, the cell of the ion, the immunoreactive cell of the invention,
the binding agent of the invention, the T cell or of the ion, or the artificial T cell
receptor of the invention.
The compositions and agents described herein are preferably capable of inducing or promoting a
cellular response, preferably cytotoxic T cell activity, against a disease characterized by
expression of CLDN6 and/or presentation of CLDN6 with class I MHC, e.g. a cancer disease.
In one aspect, the invention provides the agents and compositions described herein for use in the
methods of treatment described herein.
The ents of cancer diseases described herein can be combined with surgical resection
and/or radiation and/or ional chemotherapy.
In another aspect, the invention relates to a method for determining an immune
response in a
subject, comprising determining T cells reactive with a peptide of the invention or a cell of the
invention presenting a e of the invention or a sion product thereof in a biological
sample isolated from the subject. The method may comprise the steps of:
(a) incubating a sample comprising T cells isolated from a subject with one or more of:
(i) the peptide of the invention;
(ii) the nucleic acid of the invention comprising a nucleotide sequence encoding the peptide of
the invention; and
(iii) the cell of the invention comprising said nucleic acid or the cell of the invention presenting a
peptide of the invention or a procession product thereof;
(b) detecting the specific activation of the T cells, therefrom determining the presence or absence
of an immune response in said subject.
The invention in the above aspects of a method for ining an immune response in a t
may relate to a method for diagnosing cancer es in a subject.
In one embodiment of the methods for diagnosis, the biological sample is from a tissue or
organ
wherein the cells when the tissue or organ is disease free do not substantially express CLDN6.
Typically, the level of T cells in a biological sample is compared to a reference level, wherein a
deviation from said reference level is tive of the presence and/or stage of a disease in a
subject. The reference level may be a level as determined in a l sample (e.g., from a
healthy tissue or subject) or a median level from healthy subjects. A "deviation" from said
reference level designates any significant change, such as an increase by at least 10%, 20%, or
%, preferably by at least 40% or 50%, or even more. ably, the presence of the T cells in
said biological sample or a quantity of the T cells in the ical sample which is increased
compared to a nce level indicates the presence of a e.
T cells may be isolated from patient peripheral blood, lymph nodes, tissue samples such as
derived from biopsy and resection, or other source. Reactivity assays may be performed on
primary T cells or other appropriate derivatives. For example, T cells may be fused to generate
hybridomas. Assays for measuring T cell responsiveness are known in the art, and include
proliferation assays and ne release assays.
Assays and indices for detecting reactive T cells include but are not limited to the use of IFNy
ELISPOT and IFNy intracellular cytokine staining. Other various methods are known in the art
for ining whether a T cell clone will respond to a particuiar peptide. Typically the e
is added to a suspension of the T cells for a period of from one to three days. The se of the
T cells may be measured by proliferation, e.g., uptake of labeled thymidine, or by e of
cytokines, e.g., IL-2. Various assays are available for detecting the presence of released
cytokines. T cell cytotoxic assays can be used to detect cytotoxic T cells having specificity for
antigens. In one embodiment, cytotoxic T cells are tested for their ability to kill target cells
presenting an antigen with MHC class I molecules. Target cells presenting an antigen may be
labeled and added to a suspension of T cells from a patient sample. The cytotoxicity may be
measured by quantifying the release of label from lysed cells. Controls for spontaneous and total
release may be included in the assay.
In one embodiment of the invention, a cancer described herein involves cancer cells expressing
CLDN6 and/or presenting CLDN6 in the t of MHC molecules. In one embodiment of the
invention, diseased cells are cancer cells. In one embodiment, diseased cells such as cancer cells
are cells expressing CLDN6 and/or presenting CLDN6 in the context of MHC molecules. In one
ment, expression of CLDN6 is on the surface of a diseased cell,
In one embodiment of the invention, a cancer is selected from the group consisting of urinary
bladder cancer, ovarian cancer, in particular ovarian adenocarcinoma and ovarian
carcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung
cancer (NSCLC), in ular squamous cell lung carcinoma and arcinoma, gastric
cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell
carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in
particular malignant pleomorphic adenoma, sarcoma, in particular synovial sarcoma and
carcinosarcoma, bile duct cancer, cancer of the urinary bladder, in particular transitional cell
oma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma including
clear cell renal cell carcinoma and ary renal cell carcinoma, colon cancer, small bowel
cancer, including cancer of the ileum, in particular small bowel adenocarcinoma and
adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma,
cervical cancer, ular cancer, in particular testicular seminoma, testicular teratoma and
embryonic testicular cancer, uterine , germ cell tumors such as a teratocarcinoma or an
embryonal carcinoma, in ular germ cell tumors of the testis, and the metastatic forms
thereof.
In one embodiment of the ion, cancer cells are cancer cells of a cancer ed from the
group ting of urinary bladder cancer, ovarian cancer, in particular ovarian adenocarcinoma
and n teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-
small cell lung cancer ), in particular squamous cell lung oma and
adenocarcinoma, gastric cancer, breast cancer, c cancer, pancreatic , skin cancer, in
particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and
neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in particular synovial
sarcoma and carcincsarcoma, bile duct cancer, cancer of the urinary bladder, in particular
transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell
carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon
cancer, small bowel cancer, including cancer of the ileum, in particular small bowel
adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental
carcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular
teratoma and embryonic testicular cancer, uterine cancer, germ cell tumors such as a
teratocarcinoma or an embryonal carcinoma, in particular germ cell tumors of the , and the
metastatic forms thereof.
According to the invention, CLDN6 preferably has the amino acid sequence according to SEQ
ID NO: 1 or 2.
Other features and advantages of the instant invention will be apparent from the following
ed description and claims.
ed description of the invention
Although the present invention is bed in detail below, it is to be tood that this
invention is not limited to the particular methodologies, protocols and reagents described herein
as these may vary. It is also to be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless defined otherwise, all
technical and ific terms used herein have the same meanings as commonly understood by
one of ordinary skill in the art.
In the following, the elements of the present invention will be described. These elements are
listed with specific embodiments, however, it should be understood that they may be combined
in any manner and in any number to create additional embodiments. The variously described
examples and preferred ments should not be construed to limit the present invention to
only the explicitly described embodiments. This description should be understood to support and
encompass embodiments which combine the explicitly described ments with any number
of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of
all described elements in this application should be considered disclosed by the description of the
present application unless the context indicates ise.
Preferably, the terms used herein are defined as described in "A multilingual glossary of
biotechnological terms: (IUPAC Recommendations)", H.G.W. erger, B. Nagel, and H.
Kolbl, Eds, (1995) Helvetica a Acta, CPI-4010 Basel, Switzerland.
The ce of the present invention will employ, unless otherwise ted, conventional
methods of biochemistry, cell y, immunology, and recombinant DNA techniques which
are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual,
2'1d Edition, J. Sambrook et al. eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
1989).
Throughout this specification and the claims which follow, unless the context requires otherwise,
the word "comprise”, and variations such as "comprises" and ising”, will be understood to
imply the inclusion of a stated member, integer or step or group of members, integers or steps
but not the ion of any other member, integer or step or group of members, integers or steps
although in some embodiments such other member, integer or step or group of members,
integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated
, integer or step or group of s, integers or steps. The terms "a" and "an" and "the"
and similar reference used in the context of describing the invention (especially in the context of
the claims) are to be construed to cover both the singular and the plural, unless otherwise
indicated herein or clearly contradicted by context. Recitation of ranges of values herein is
merely intended to serve as a shorthand method of referring individually to each separate value
falling within the range. Unless ise indicated herein, each individual value is incorporated
into the specification as if it were individually recited herein.
All s described herein can be performed in any le order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any and all examples, or
exemplary ge (e.g., "such as"), provided herein is intended merely to better illustrate the
invention and does not pose a limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any non-claimed element
essential to the practice of the invention.
Several documents are cited throughout the text of this specification. Each of the documents
cited herein (including all patents, patent applications, scientific publications, manufacturer's
specifications, instructions, etc), whether supra or infia, are hereby incorporated by reference in
their ty. Nothing herein is to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior ion.
The term binant” in the context of the present invention means ”made throu h
g genetic
engineering". ably, a binant object" such as a recombinant cell in the context of the
present invention is not occurring naturally.
The term "naturally occurring" as used herein refers to the fact that an object can be found in
nature. For example, a peptide or nucleic acid that is present in an organism (including viruses)
and can be isolated from a source in nature and which has not been intentionally modified by
man in the laboratory is naturally occurring.
The term ”immune se" refers to an integrated bodily response to an n and preferably
refers to a cellular immune response or a cellular as well as a humoral immune
response. The
immune se may be tive/preventive/prophylactic and/or therapeutic.
"Inducing an immune response" may mean that there was no immune response against a
particular antigen before induction, but it may also mean that there was a certain level of immune
response against a particular antigen before induction and after induction said immune response
is enhanced. Thus, "inducing an immune response" also includes "enhancing an immune
response". Preferably, after inducing an immune response in a subject, said subject is protected
from developing a disease such as a cancer disease or the disease condition is ameliorated by
inducing an immune response. For example, an immune response against a associated
antigen such as CLDN6 may be induced in a patient having a cancer disease or in a subject being
at risk of developing a cancer disease. ng an immune response in this case may mean that
the e condition of the subject is ameliorated, that the subject does not develop metastases,
or that the t being at risk of developing a cancer disease does not develop a cancer disease.
A "cellular immune response", a "cellular response", a "cellular response against an antigen" or a
similar term is meant to include a cellular response directed to cells terized by presentation
of an antigen with class I or class ll MHC. The cellular se relates to cells called T cells or
hocytes which act as either 'helpers' or 'killers'. The helper T cells (also termed CD4+ T
cells) play a central role by regulating the immune response and the killer cells (also termed
cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells,
preventing the production of more diseased cells.
The term "antigen" relates to an agent comprising an epitope against which an immune
response
is to be ted and/or is directed. Preferably, an antigen in the context of the present invention
is a le which, optionally after processing, induces an immune reaction, which is
preferably specific for the antigen or cells expressing and/or presenting the antigen. The term
"antigen" includes in particular proteins and es. An antigen is preferably a product which
corresponds to or is derived from a naturally occurring antigen. Such naturally occurring
antigens may include or may be derived from tumor-associated antigens.
In particular, the antigen or peptide fragments thereof should be recognizable by a T cell
receptor. Preferably, the antigen or e if recognized by a T cell receptor is able to induce in
the presence of appropriate co-stimulatory signals, clonal expansion of the T cell carrying the T
cell receptor recognizing the antigen or peptide. In the context of the embodiments of the present
invention, the antigen is preferably presented by a cell, ably by an antigen presenting cell
and/or a diseased cell, in the context of MHC molecules, which may result in an immune
reaction against the antigen (or cell presenting the antigen).
In a preferred embodiment, an antigen is a tumor—associated antigen, i.e., a constituent of cancer
cells which may be derived from the cytoplasm, the cell surface and the cell nucleus, in
particular those antigens which are produced, ably in large quantity, intracellular or as
surface antigens on cancer cells.
In the context of the present ion, the term ”tumor-associated antigen" or "tumor antigen"
relates to prctei .s that re under normal ions specifically expressed in a limited number of
tissues and/or organs or in specific developmental , for example, the tumor—associated
antigen may be under normal ions specifically expressed in stomach tissue, preferably in
the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta,
or in germ line cells, and are expressed or aberrantly sed in one or more tumor or cancer
tissues. In this context, "a d number" ably means not more than 3, more preferably
not more than 2. The tumor-associated antigens in the t of the present invention include,
for example, differentiation antigens, ably cell type specific differentiation antigens, i.e.,
proteins that are under normal conditions specifically sed in a certain cell type at a n
differentiation stage, cancer/testis antigens, i.e., proteins that are under normal conditions
specifically expressed in testis and sometimes in placenta, and germ line specific ns. In the
context of the present invention, the tumor-associated antigen is preferably associated with the
cell surface of a cancer cell and is preferably not or only rarely expressed in normal tissues.
Preferably, the tumor-associated antigen or the aberrant expression of the tumor—associated
antigen identifies cancer cells. In the context of the t invention, the tumor-associated
n that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer
disease, is preferably a self—protein in said subject. In preferred embodiments, the tumor-
associated antigen in the context of the present invention is expressed under normal conditions
specifically in a tissue or organ that is non—essential, i.e., tissues or organs which when damaged
by the immune system do not lead to death of the subject, or in organs or structures of the body
which are not or only hardly ible by the immune system. ably, the amino acid
sequence of the tumor-associated antigen is identical between the associated antigen
which is expressed in normal tissues and the tumor-associated antigen which is expressed in
cancer tissues. Preferably, a tumor-associated antigen is presented by a cancer cell in which it is
expressed.
Various aspects of the ion involve the tumor—associated antigen CLDN6 and the present
ion may involve the stimulation or provision of an anti-tumor CTL reaction against cancer
cells expressing said tumor-associated n and preferably presenting said tumor-associated
antigen with class I MHC.
Claudins are a family of proteins that are the most important components of tight ons,
where they establish the paracellular barrier that controls the flow of molecules in the
intercellular space between cells of an epithelium. Claudins are transmembrane proteins
ng the membrane 4 times with the inal and the C-terminal end both located in the
cytoplasm. The first extracellular loop, termed ECl or ECLl, consists on average of 53 amino
acids, and the second extracellular loop, termed EC2 or ECL2, ts of around 24 amino
acids. Cell surface proteins of the claudin family, such as CLDN6, are sed in tumors of
various origins, and are particularly suited as target structures in tion with antibody-
mediated cancer immunotherapy due to their selective expression (no expression in a toxicity
relevant normal tissue) and localization to the plasma ne.
CLDN6 has been identified as differentially expressed in tumor tissues, with the only normal
tissues expressing CLDN6 being placenta.
CLDN6 has been found to be expressed, for example, in ovarian cancer, lung cancer, gastric
cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, melanomas, head neck
cancer, sarcomas, bile duct cancer, renal cell cancer, and urinary bladder cancer. CLDN6 is a
particularly red target for the prevention and/or treatment of ovarian cancer, in particular
n adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung
cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung
carcinoma and adenocarcinoma, gastric cancer, breast cancer, hepatic , pancreatic
cancer,
skin , in particular basal cell oma and squamous cell oma, malignant
melanoma, head and neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in
particular synovial a and carcinosarcoma, bile duct cancer, cancer of the urinary bladder,
in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in ular
renal cell carcinoma ing clear cell renal cell carcinoma and papillary renal cell carcinoma,
colon cancer, small bowel cancer, including cancer of the ileum, in particular small bowel
adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental
choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular
ma and embryonic testicular cancer, uterine cancer, germ cell tumors such as a
teratocarcinoma or an embryonal carcinoma, in particular germ cell tumors of the testis, and the
metastatic forms thereof. In one embodiment, the cancer disease associated with CLDN6
expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic
ovarian cancer and atic lung cancer. Preferably, the ovarian cancer is a carcinoma or an
adenocarcinoma. Preferably, the lung cancer is a carcinoma or an adenocarcinoma, and
preferably is bronchiolar cancer such as a bronchiolar carcinoma or bronchiolar adenocarcinoma.
The term "CLDN" as used herein means claudin and includes CLDN6. ably, a claudin is
human claudin. The term "CLDN6" relates to claudin 6 and includes
any variants thereof.
The term "CLDN6" preferably relates to human CLDN6, and, in particular, to a n
comprising, preferably consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:
2 of the sequence listing or a t of said amino acid sequence. The first extracellular loop of
CLDN6 preferably comprises amino acids 28 to 80, more preferably amino acids 28 to 76 of the
amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO:
2. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160,
preferably amino acids 141 to 159, more preferably amino acids 145 to 157 of the amino acid
sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2. Said
first and second extracellular loops preferably form the extracellular portion of CLDN6.
The term "varian " according to the invention , in particular, to s, splice variants,
conformations, isoforrns, allelic variants, species variants and species homologs, in particular
those which are naturally present. An allelic variant relates to an alteration in the normal
sequence of a gene, the significance of which is often unclear. Complete gene sequencing often
identifies numerous allelic variants for a given gene. A species homolog is a nucleic acid or
amino acid sequence with a different species of origin from that of a given nucleic acid or amino
acid sequence. The term "variant" shall encompass any anslationally modified variants and
conformation variants.
According to the various aspects of the invention, the aim is preferably to induce or determine an
immune response against cancer cells sing CLDN6 and preferably being characterized by
presentation of CLDN6, and to diagnose, treat or prevent a cancer disease ing cells
expressing CLDN6. Preferably the immune response involves the stimulation of an anti-CLDN6
CTL response t cancer cells expressing CLDN6 and preferably presenting CLDN6 with
class I MHC.
ing to the ion, the term "CLDN6 positive cancer" or similar terms means a cancer
involving cancer cells expressing CLDN6, preferably on the e of said cancer cells.
Alternatively or onally, said cancer cells expressing CLDN6 present CLDN6 in the context
of MHC molecules. Cancer cells presenting CLDN6 in the context of MHC molecules can be
targeted by immunoreactive cells carrying T cell receptors while cancer cells expressing CLDN6
on the surface can be targeted by immunoreactive cells carrying artificial T cell receptors.
"Cell surface" is used in accordance with its normal meaning in the art, and thus es the
outside of the cell which is accessible to binding by proteins and other molecules
CLDN6 is expressed on the e of cells if it is located at the surface of said cells and is
accessible to binding by CLDN6-specific antibodies added to the cells.
The term "extracellular portion" or "exodomain" in the context of the present ion refers to
a part of a molecule such as a protein that is facing the ellular space of a cell and
preferably is accessible from the outside of said cell, e.g., by antigen-binding molecules such as
antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops
or domains or a fragment thereof.
The term on" refers to a on. With respect to a particular structure such as an amino
acid sequence or protein the term "portion" thereof may designate a continuous or a
discontinuous on of said structure. Preferably, a portion of an amino acid sequence
comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, ably at least
40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even
more preferably at least 80%, and most preferably at least 90% of the amino acids of said amino
acid sequence. Preferably, if the portion is a discontinuous fraction said discontinuous fraction is
composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure, each part being a continuous element
of the structure. For example, a discontinuous fraction of an amino acid sequence may be
composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably not more than 4 parts of said amino acid
ce, wherein each part preferably comprises at least 5 continuous amino acids, at least 10
uous amino acids, preferably at least 20 continuous amino acids, preferably at least 30
uous amino acids of the amino acid sequence.
The terms "pa H and "fragment" are used interchangeably herein and refer to a continuous
t. For example, a part of a structure such as an amino acid sequence or protein refers to a
continuous element of said structure. A portion, a part or a fragment of a structure preferably
comprises one or more functional properties of said structure. For example, a portion, a part or a
fragment of an epitope, peptide or protein is preferably immunologically lent to the
epitope, peptide or protein it is derived from. In the context of the present invention, a "pa " of
structure such as an amino acid sequence preferably comprises, preferably consists of at least
%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least
99% of the entire structure or amino acid sequence. A part or nt of a protein
sequence
preferably comprises a sequence of at least 6, in ular at least 8, at least 12, at least 15, at
least 20, at least 30, at least 50, or at least 100 consecutive amino acids of the protein
sequence.
Portions, parts or nts as discussed above are encompassed by the term "variant" used
herein.
According to the invention, CLDN6 is not substantially expressed in a cell if the level of
expression is lower compared to expression in placenta cells or placenta tissue. Preferably, the
level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05%
of the expression in placenta cells or placenta tissue or even lower. Preferably, CLDN6 is not
substantially expressed in a cell if the level of expression exceeds the level of expression in non—
cancerous tissue other than placenta by no more than 2-fold, preferably 15-fold, and preferably
does not exceed the level of expression in said non-cancerous . Preferably, CLDN6 is not
substantially expressed in a cell if the level of expression is below the ion limit and/or if
the level of expression is too low to allow binding by specific antibodies added to the
cells.
According to the invention, CLDN6 is expressed in a cell if the level of expression exceeds the
level of sion in non-cancerous tissue other than placenta preferably by more than 2-fold,
preferably 10-fold, 100-fold, IOOO-fold, or lOOOO-fold. Preferably, CLDN6 is expressed in a cell
if the level of sion is above the detection limit and/or if the level of sion is high
enough to allow binding by CLDN6-specific antibodies added to the cells. Preferably, CLDN6
expressed in a cell is expressed or exposed on the e of said cell.
"Target cell" shall mean a cell which is a target for an immune response such as a cellular
immune response. Target cells include cells that present an antigen or an antigen epitope, i.e. a
peptide fragment derived from an antigen, and include any undesirable cell such as a cancer cell.
In preferred embodiments, the target cell is a cell expressing CLDN6 which preferably is present
on the cell surface and/or presented with class I MHC.
The term "epitope" refers to an nic determinant in a molecule such as an antigen, i.e., to a
part in or fragment of the molecule that is recognized by the immune system, for example, that is
recognized by a T cell, in particular when presented in the context of MHC molecules. An
epitope of a protein such as a tumor-associated antigen preferably comprises a continuous or
discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5
and 50, more preferably between 8 and 30, most ably between 10 and 25 amino acids in
length, for example, the epitope may be preferably 9, 10, ll, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the
t of the present invention is a T cell epitope.
Terms such as "epitope", " antigen fragment", en peptide" or "immunogenic peptide" are
used interchangeably herein and preferably relate to an incomplete representation of an n
which is preferably e of ing an immune response against the antigen or a cell
expressing or comprising and preferably presenting the antigen. Preferably, the terms relate to an
WO 50327
immunogenic portion of an antigen. ably, it is a portion of an antigen that is recognized
(i.e., cally bound) by a T cell receptor, in ular if presented in the context of MHC
molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule
such as on the surface of a cell and thus are MHC binding peptides. As used , a peptide is
said to “bind to" an MHC class I or class II molecule if such binding is able using any
assay known in the art.
Preferably, the es disclosed herein comprising an amino acid sequence selected from the
group consisting of SEQ ID NOS: 3, 4 and 5 or a variant of said amino acid ce are e
of stimulating an immune response, preferably a cellular response t CLDN6 or cells
characterized by expression of CLDN6 and ably characterized by presentation of CLDN6.
Preferably, such peptide is capable of ating a cellular response against a cell characterized
by presentation of CLDN6 with class I MHC and preferably is capable of stimulating CLDN6-
responsive CTL. Preferably, the peptides according to the invention are MHC class I and/or class
II presented peptides or can be processed to produce MHC Class I and/or class II presented
peptides. Preferably, the sequence bound to the MHC molecule is selected from SEQ ID NOS: 3,
4 and 5.
If an antigen peptide is to be presented directly, i.e. without processing, in particular without
cleavage, it has a length which is le for binding to an MHC molecule, in particular a class I
MHC molecule, and preferably is 7—20 amino acids in length, more preferably 7-12 amino acids
in length, more preferably 8-11 amino acids in length, in particular 9 or 10 amino acids in length.
Preferably the sequence of an antigen peptide which is to be presented directly substantially
corresponds and is preferably completely identical to a sequence selected from SEQ ID NOs: 3,
4 and 5.
If an antigen peptide is to be presented following processing, in particular following cleavage,
the peptide produced by processing has a length which is suitable for binding to an MHC
molecule, in particular a class I MHC molecule, and preferably is 7-20 amino acids in length,
more preferably 7-12 amino acids in , more preferably 8-11 amino acids in length, in
particular 9 or 10 amino acids in length. Preferably, the sequence of the peptide which is to be
presented following processing substantially ponds and is preferably completely identical
to a sequence selected from SEQ ID NOs: 3, 4 and 5. Thus, an antigen peptide according to the
invention in one embodiment comprises a sequence selected from SEQ ID NOs: 3, 4 and 5 and
2015/056899
following processing of the n peptide makes up a sequence selected from SEQ ID NOS: 3,
4 and 5.
Peptides having amino acid sequences substantially corresponding to a ce of a peptide
which is presented by MHC les may differ at one or more residues that are not essential
for TCR recognition of the peptide as presented by the MHC, or for peptide binding to MHC.
Such substantially corresponding es preferably are also capable of stimulating an antigen-
c cellular response such as antigen-specific CTL. Peptides having amino acid sequences
differing from a presented peptide at residues that do not affect TCR recognition but improve the
stability of binding to MHC may improve the immunogenicity of the antigen peptide, and may
be referred to herein as "optimized peptides". Using existing knowledge about which of these
residues may be more likely to affect binding either to the MHC or to the TCR, a rational
ch to the design of substantially corresponding peptides may be employed. Resulting
peptides that are functional are contemplated as antigen peptides. ces as discussed above
are encompassed by the term "variant" used herein.
"Antigen processing" refers to the degradation of an antigen into procession products, which are
fragments of said antigen (cg, the degradation of a protein into peptides) and the association of
one or more of these fragments (e.g., via binding) with MHC molecules for tation by cells,
preferably antigen presenting cells to specific T cells.
An antigen-presenting cell (APC) is a cell that displays antigen in the context of major
histocompatibility complex (MHC) on its surface. T cells may recognize this x using their
T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells.
Professional antigen-presenting cells are very efficient at internalizing antigen, either by
phagocytosis or by receptor—mediated endocytosis, and then ying a fragment of the antigen,
bound to a class II MHC molecule, on their membrane. The T cell recognizes and interacts with
the antigen-class II MHC molecule x on the membrane of the antigen-presenting cell. An
additional mulatory signal is then produced by the antigen—presenting cell, leading to
activation of the T cell. The expression of co-stimulatory molecules is a g feature of
professional antigen-presenting cells. Antigen—presenting cells include professional antigen-
presenting cells and non-professional antigen-presenting cells.
The main types of professional antigen—presenting cells are dendritic cells, which have the
broadest range of antigen presentation, and are ly the most important antigen-presenting
cells, macrophages, B-cells, and certain ted epithelial cells.
Non-professional antigen-presenting cells do not constitutively express the MHC class 11
proteins required for interaction with naive T cells; these are expressed only upon ation of
the non—professional antigen-presenting cells by certain cytokines such as IFNy.
Dendritic cells (DCs) are leukocyte populations that present antigens captured in peripheral
tissues to T cells via both MHC class II and 1 antigen presentation. pathways. It is well known
that tic cells are potent inducers of immune responses and the activation of these cells is a
critical step for the induction of antitumoral immunity.
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-
infiltrating cells, peritumoral tissues—infiltrating cells, lymph nodes, spleen, skin, umbilical cord
blood or any other suitable tissue or fluid. For example, dendritic cells may be entiated ex
vivo by adding a combination of cytokines such as GM-CSF, lL-4, IL-13 and/or TNFa to
cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells
ted from peripheral blood, umbilical cord blood or bone marrow may be differentiated into
dendritic cells by adding to the culture medium combinations of GM-CSF, lL—3, TNFu, CD40
ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and
proliferation of dendritic cells.
Dendritic cells are conveniently categorized as "immature" and "mature" cells, which can be
used as a simple way to discriminate between two well characterized phenotypes. However, this
nomenclature should not be construed to exclude all possible intermediate stages of
differentiation.
re dendritic cells are characterized as antigen presenting cells with a high ty for
antigen uptake and processing, which correlates with the high expression of Fey receptor and
mannose receptor. The mature phenotype is typically characterized by a lower expression of
these markers, but a high expression of cell surface molecules responsible for T cell activation
such as class I and class II MHC, on les (e. g. CD54 and CD11) and ulatory
molecules (e. g., CD40, CD80, CD86 and 4-1 BB).
2015/056899
Dendritic cell maturation is referred to as the status of dendritic cell activation at which such
antigen-presenting dendritic cells lead to T cell priming, while presentation by immature
dendritic cells results in tolerance. Dendritic cell maturation is chiefly caused by ecules
with microbial features detected by innate receptors (bacterial DNA, viral RNA, endotoxin, etc),
pro-inflammatory cytokines (TNF, IL—l, IFNs), ligation of CD40 on the dendritic cell surface by
CD40L, and substances released from cells oing stressful cell death. The dendritic cells
can be derived by culturing bone marrow cells in vitra with cytokines, such as granulocyte-
macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.
Cells such as antigen presenting cells or target cells can be loaded with MHC class I presented
peptides by exposing, i.e. pulsing, the cells with the e or transducing the cells with nucleic
acid, preferably RNA, encoding a e or protein comprising the peptide to be presented, e. g.
a nucleic acid encoding the antigen.
In some embodiments, a pharmaceutical composition of the invention comprises an antigen
presenting cell loaded with antigen peptide. In this respect, protocols may rely on in vitro
culture/differentiation of dendritic cells manipulated in such a way that they artificially t
n peptide. Production of genetically engineered dendritic cells may involve introduction of
nucleic acids encoding antigens or antigen peptides into dendritic cells. Transfection of dendritic
cells with mRNA is a promising antigen-loading technique of stimulating strong antitumor
immunity. Such transfection may take place ex viva, and a pharmaceutical composition
comprising such transfected cells may then be used for eutic purposes. Alternatively, a
gene delivery vehicle that targets a dendritic or other antigen presenting cell may be
stered to a patient, resulting in transfection that occurs in viva. In viva and ex viva
ection of dendritic cells, for example, may generally be performed using any methods
known in the art, such as those described in WO 97/24447, or the gene gun approach described
by Mahvi et al., Immunology and cell y 75: 456—460,]997. Antigen g of dendritic
cells may be achieved by incubating tic cells or progenitor cells with n, DNA (naked
or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacteria or viruses
(e.g., vaccinia, fowipox, adenovirus or lentivirus vectors).
The term "immunogenicity" s to the relative efficiency of an antigen to induce an immune
reaction.
The term "immune effector functions" in the context of the present invention includes any
functions mediated by components of the immune system that result, for example, in the killing
of tumor cells, or in the inhibition of tumor growth and/or inhibition of tumor development,
including inhibition of tumor dissemination and metastasis. Preferably, the immune effector
functions in the context of the present invention are T cell mediated effector functions. Such
ons comprise in the case of a helper T cell (CD4+ 1 cell) the recognition of an antigen or an
antigen peptide d from an antigen in the context of MHC class II molecules by T cell
receptors, the release of cytokines and/or the activation of CD8+ lymphocytes (CTLs) and/or B-
cells, and in the case of CTL the recognition of an antigen or an antigen peptide derived from an
antigen in the context of MHC class I molecules by T cell ors, the ation of cells
presented in the context of MHC class I molecules, i.e., cells characterized by presentation of an
antigen with class I MHC, for example, via apoptosis or perform-mediated cell lysis, production
of cytokines such as IFN-y and , and specific cytolytic killing of antigen expressing target
cells.
The term oreactive cell" or ”immune effector cell" in the context of the present invention
relates to a cell which exerts effector functions during an immune reaction. An “immunoreactive
cell" preferably is capable of binding an antigen such as an antigen expressed on the surface of a
cell or a cell characterized by presentation of an antigen or an n peptide derived from an
antigen and mediating an immune response. For example, such cells secrete cytokines and/or
chemokines, kill microbes, secrete antibodies, recognize infected or cancerous cells, and
optionally ate such cells. For example, immunoreactive cells comprise T cells (cytotoxic T
cells, helper T cells, tumor infiltrating T , B cells, natural killer cells, neutrophils,
macrophages, and dendritic cells. Preferably, in the context of the t invention,
"immunoreactive cells" are T cells, preferably CD4+ and/or CD8+ T cells.
Preferably, an oreactive cell" recognizes an antigen or an antigen peptide d from
an antigen with some degree of specificity, in particular if presented in the context of MHC
molecules such as on the surface of antigen presenting cells or diseased cells such as cancer
cells. Preferably, said recognition s the cell that recognizes an antigen or an antigen
peptide derived from said antigen to be sive or reactive. 1f the cell is a helper T cell (CD4+
T cell) bearing receptors that recognize an n or an antigen peptide derived from an antigen
in the context of MHC class II les such responsiveness or reactivity may involve the
2015/056899
e of cytokines and/or the activation of CD8+ lymphocytes (CTLs) and/or B—cells. If the cell
is a CTL such responsiveness or vity may involve the elimination of cells presented in the
context of MHC class I molecules, i.e., cells characterized by presentation of an antigen with
class I MHC, for example, via apoptosis or perform-mediated cell lysis. According to the
invention, CTL responsiveness may include sustained calcium flux, cell division, production of
cytokines such as IFN-y and TNF—ct, ulation of activation s such as CD44 and
CD69, and specific tic killing of n expressing target cells. CTL responsiveness may
also be determined using an artificial er that accurately indicates CTL responsiveness.
Such CTL that recognizes an antigen or an antigen peptide derived from an antigen and are
responsive or reactive are also termed "antigen-responsive CTL" herein. If the cell is a B cell
such responsiveness may e the release of immunoglobulins.
According to the invention, the term "immunoreactive cell" also includes a cell which can
mature into an immune cell (such as T cell, in ular T helper cell, or cytolytic T cell) with
suitable stimulation. Immunoreactive cells comprise CD34+ hematopoietic stem cells, immature
and mature T cells and immature and mature B cells. If production of cytolytic or T helper cells
recognizing an antigen is desired, the immunoreactive cell is contacted with a cell presenting an
antigen or antigen peptide under conditions which favor production, differentiation and/or
selection of cytolytic T cells and of T helper cells. The differentiation of T cell precursors into a
cytolytic T cell, when exposed to an antigen, is similar to clonal selection of the immune system.
A ”lymphoid cell” is a cell which, optionally after le ation, e.g. after transfer of a T
cell receptor, is capable of producing an immune response such as a cellular immune response,
or a precursor cell of such cell, and includes lymphocytes, preferably T lymphocytes,
lymphoblasts, and plasma cells. A lymphoid cell may be an immunoreactive cell as described
herein. A preferred lymphoid cell is a T cell lacking endogenous expression of a T cell receptor
and which can be modified to express such T cell receptor on the cell surface.
The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper
cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T
cells.
T cells belong to a group of white blood cells known as lymphocytes, and play a central role in
cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells
WO 50327 2015/056899
and natural killer cells by the presence of a special receptor on their cell surface called T cell
receptors (TCR). The thymus is the principal organ responsible for the T cell‘s maturation of T
cells. Several different subsets of T cells have been ered, each with a ct function.
T helper cells assist other white blood cells in immunologic processes, including maturation of B
cells into plasma cells and activation of xic T cells and hages, among other
functions. These cells are also known as CD4+ T cells because they
express the CD4 protein on
their surface. Helper T cells become activated when they are presented with peptide antigens by
MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs).
Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or
assist in the active immune response.
Cytotoxic T cells destroy vitally infected cells and tumor cells, and are also implicated in
transplant rejection. These cells are also known as CD8+ T cells since they express the CD8
glycoprotein at their surface. These cells recognize their targets by binding to antigen associated
with MHC class I, which is present on the surface of nearly every cell of the body.
A majority of T cells have a T cell or (TCR) existing as a complex of several proteins. The
actual T cell receptor is composed of two separate peptide chains, which are produced from the
independent T cell receptor alpha and beta (TCRa and TCRB) genes and are called a- and B-TCR
. 75 T cells (gamma delta T cells) represent a small subset of T cells that
s a distinct
T cell receptor (TCR) on their surface. However, in 78 T cells, the TCR is made up of one y-
chain and one 8-chain. This group of T cells is much less common (2% of total T cells) than the
(x13 T cells,
The structure of the T cell receptor is very similar to immunoglobulin Fab fragments, which are
regions defined as the combined light and heavy chain of an antibody arm. Each chain of the
TCR is a member of the immunoglobulin superfamily and possesses one N-terminal
immunoglobulin (Ig)—variable (V) domain, one stant (C) domain, a transmembrane/cell
membrane-spanning region, and a short cytoplasmic tail at the C-terminal end.
According to the invention, the term ble region of a T cell receptor" relates to the variable
domains of the TCR chains.
The variable region of both the TCR (Jr-chain and B—chain have three hypervariable or
complementarity determining regions , whereas the variable region of the B-chain has an
additional area of hypervariability (HV4) that does not normally contact antigen and therefore is
not considered a CDR. CDR3 is the main CDR responsible for recognizing processed antigen,
although CDR] of the (it-chain has also been shown to ct with the N-terminal part of the
antigenic peptide, whereas CDR] of the B-chain interacts with the C-terminal part of the peptide.
CDR2 is thought to recognize the MHC. CDR4 of the B-chain is not thought to participate in
antigen recognition, but has been shown to interact with superantigens.
According to the invention, the term ”at least one of the CDR sequences" preferably means at
least the CDR3 sequence. The term "CDR sequences of a T cell, receptor chain" preferably
relates to CDR], CDRZ and CDR3 of the a-chain or B-chain of a T cell receptor.
The constant domain of the TCR domain consists of short connecting ces in which a
cysteine residue forms disulfide bonds, which forms a link between the two chains.
All T cells ate from hematopoietic stem cells in the bone marrow. Hematopoietic
progenitors derived from hematopoietic stem cells populate the thymus and expand by cell
division to generate a large tion of immature thymocytes. The earliest thymocytes express
neither CD4 nor CD8, and are therefore classed as double—negative (CD4-CD8-) cells. As they
progress through their pment they become double-positive thymocytes D8+), and
finally mature to single-positive (CD4+CD8~ or CD4-CD8+) ytes that are then released
from the thymus to peripheral tissues.
The first signal in activation of T cells is provided by binding of the T cell receptor to a short
peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures
that only a T cell with a TCR specific to that peptide is activated. The partner cell is y a
professional antigen presenting cell (AFC), usually a dendritic cell in the case of naive
responses, although B cells and hages can be important APCs. The peptides presented to
CD8+ T cells by MHC class I molecules are 8-10 amino acids in length; the peptides presented
to CD4+ T cells by MHC class II molecules are , as the ends of the g cleft of the
MHC class II molecule are open.
T cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T
cells may be present within (or isolated from) bone marrow, peripheral blood or a on of
bone marrow or peripheral blood of a mammal, such as a t, using a commercially available
cell separation . Alternatively, T cells may be derived from related or unrelated humans,
non-human animals, cell lines or cultures. A "sample comprising T cells" may, for example, be
peripheral blood mononuclear cells (PBMC).
T cells may be stimulated with antigen, peptide, nucleic acid and/or antigen ting cells
(APCs) that express an antigen. Such stimulation is performed under conditions and for a time
sufficient to permit the generation of T cells that are specific for an antigen, a peptide and/0r
cells presenting an antigen or a peptide.
Specific activation of CD4+ or CD8+ T cells may be detected in a variety of ways. Methods for
detecting specific T cell activation include detecting the proliferation of T cells, the production
of cytokines (e.g., lymphokines), or the generation of tic activity. For CD4+ T cells, a
preferred method for detecting specific T cell activation is the detection of the proliferation of T
cells. For CD8+ T cells, a preferred method for ing specific T cell activation is the
detection of the generation of cytolytic activity.
In order to generate CD8+ T cell lines, antigen—presenting cells, preferably autologous antigen-
presenting cells, transfected with a nucleic acid which produces the antigen may be used as
stimulator cells.
Nucleic acids such as RNA encoding T cell receptor (TCR) chains may be introduced into
lymphoid cells such as T cells or other cells with lytic ial. In a le embodiment, the
TCR (1- and ns are cloned out from an antigen-specific T cell line and used for adoptive T
cell y. In this respect, the present invention provides T cell receptors specific for CLDN6
or CLDN6 peptides disclosed herein. In general, this aspect of the invention relates to T cell
receptors which recognize or bind CLDN6 peptides presented in the context of MHC. The
nucleic acids encoding u- and B-chains of a T cell or, e.g. a T cell or provided
according to the present invention, may be contained on separate nucleic acid molecules such as
expression vectors or alternatively, on a single c acid molecule. Accordingly, the term "a
c acid encoding a T cell receptor" or similar terms relate to nucleic acid molecules
encoding the T cell receptor chains on the same or preferably on different nucleic acid
molecules.
The term "immunoreactive cell ve with a peptide" relates to an immunoreactive cell which
when it recognizes the peptide, in particular if ted in the context of MHC molecules such
as on the surface of antigen presenting cells or diseased cells such as cancer cells, exerts effector
functions of immunoreactive cells as described above.
The term "T cell receptor reactive with a peptide" relates to a T cell receptor which when present
on an immunoreactive cell recognizes the peptide, in particular if presented in the context of
MHC molecules such as on the surface of antigen ting cells or diseased cells such as
cancer cells, such that the immunoreactive cell exerts or functions of immunoreactive cells
as described above.
The term "antigen-reactive T cell" or similar terms relate to a T cell which recognizes an antigen
if ted in the context of MHC molecules such as on the surface of antigen presenting cells
or diseased cells such as cancer cells and exerts or functions of T cells as described above.
The term "antigen-specifc id cell" relates to a lymphoid cell which, in particular when
provided with an antigen-specific T cell receptor, recognizes the antigen if presented in the
context of MHC molecules such as on the surface of antigen presenting cells or diseased cells
such as cancer cells and preferably exerts effector ons of T cells as described above. T cells
and other lymphoid cells are considered to be specific for antigen if the cells kill target cells
expressing an antigen and/or presenting an antigen peptide. T cell city may be evaluated
using any of a variety of standard techniques, for example, within a chromium release assay or
proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be
measured
The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I
and MHC class II molecules and relate to a complex of genes which occurs in all rates.
MHC proteins or molecules are important for signaling between lymphocytes and antigen
presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules
bind peptides and present them for recognition by T cell ors. The proteins encoded by the
MHC are expressed on the surface of cells, and display both self antigens (peptide fragments
from the cell itself) and nonself antigens (e.g., fragments of invading microorganisms) to a T
cell.
The MHC region is divided into three subgroups, class 1, class II, and class III. MHC class 1
proteins contain an (it—chain and BZ-microglobulin (not part of the MHC encoded by chromosome
). They present antigen fragments to cytotoxic T cells. On most immune system cells,
specifically on antigen-presenting cells, MHC class 11 proteins contain (1— and B—chains and they
present n fragments to T-helper cells. MHC class III region encodes for other immune
components, such as complement components and some that encode cytokines.
In humans, genes in the MHC region that encode antigen—presenting proteins on the cell surface
are referred to as human leukocyte antigen (HLA) genes. However the abbreviation MHC is
often used to refer to HLA gene ts. HLA genes include the nine led classical MHC
genes: HLA-A, HLA-B, HLA~C, HLA—DPAl, HLA-DPBl, HLA—DQAI, HLA-DQBl, HLA-
DRA, and BI.
In one preferred embodiment of all aspects of the invention an MHC molecule is an HLA
molecule.
By "cell characterized by presentation of an antigen", "cell presenting an antigen", "antigen
presented by a cell", "antigen presented" or similar expressions is meant a cell such as a ed
cell such as a cancer cell, or an antigen presenting cell presenting the antigen it expresses or a
fragment derived from said antigen, e.g. by processing of the antigen, in the context of MHC
les, in ular MHC Class I molecules. Similarly, the terms "disease terized by
presentation of an antigen" denotes a e involving cells characterized by presentation of an
antigen, in ular with class I MHC. Presentation of an antigen by a cell may be effected by
transfecting the cell with a nucleic acid such as RNA encoding the antigen.
By "fragment of an antigen which is ted" or similar expressions is meant that the fragment
can be presented by MHC class I or class 11, preferably MHC class I, e. g. when added directly to
antigen presenting cells. In one embodiment, the fragment is a fragment which is naturally
presented by cells expressing an n.
Some therapeutic methods are based on a reaction of the immune system of a patient, which
WO 50327
results in a lysis of diseased cells which t an antigen with class I MHC. In this connection,
for example autologous cytotoxic T lymphocytes specific for a complex of an antigen peptide
and an MHC molecule may be administered to a patient having a disease. The production of such
cytotoxic T lymphocytes in vitro is known. An example of a method of differentiating T cells
can be found in WO—A-9633265. Generally, a sample containing cells such as blood cells is
taken from the patient and the cells are ted with a cell which presents the complex and
which can cause propagation of cytotoxic T lymphocytes (e.g. dendritic cells). The target cell
may be a ected cell such as a COS cell. These transfected cells present the desired complex
on their surface and, when contacted with cytotoxic T lymphocytes, stimulate propagation of the
latter. The clonally expanded autologous cytotoxic T lymphocytes are then administered to the
patient.
In another method of selecting cytotoxic T lymphocytes, fluorogenic tetramers of MHC class I
molecule/peptide complexes are used for obtaining specific clones of cytotoxic T lymphocytes
(Altman et al.(1996), e -96; Dunbar et a1. (1998), Curr. Biol. 8:413-416, 1998).
Furthermore, cells presenting the desired x (e.g. dendritic cells) may be combined with
cytotoxic T lymphocytes of healthy individuals or another species (e.g. mouse) which may result
in propagation of specific cytotoxic T lymphocytes with high affinity. The high affinity T cell
receptor of these propagated specific T lymphocytes may be cloned and optionally humanized to
a ent extent, and the T cell receptors thus obtained then transduced via gene transfer, for
e using retroviral vectors, into T cells of patients. Adoptive transfer may then be carried
out using these genetically altered T lymphocytes (Stanislawski et al.(2001), Nat Immunol.
22962-70; Kessels et a1. (2001), Nat Immunol. 2:957-61.
Cytotoxic T cytes may also be ted in vivo in a manner known per se. One method
uses nonproliferative cells expressing an MHC class I/peptide complex. The cells used here will
be those which usually express the complex, such as irradiated tumor cells or cells transfected
with one or both genes necessary for presentation of the complex (i.e. the antigenic peptide and
the presenting MHC molecule). Another preferred form is the introduction of an antigen in the
form of inant RNA which may be introduced into cells by liposomal er or by
electroporation, for example. The resulting cells present the complex of interest and are
recognized by autologous cytotoxic T lymphocytes which then propagate.
A similar effect can be achieved by combining an antigen or an anti
gen peptide with an adjuvant
in order to make incorporation into antigen-presenting cells in viva possible. The antigen or
n peptide may be represented as protein, as DNA (e.g. within a vector) or as RNA. The
antigen may be processed to produce a peptide r for the MHC. molecule, while a fragment
thereof may be ted without the need for r processing. The latter is the case in
particular, if these can bind to MHC les. Preference is given to administration forms in
which the complete antigen is processed in vivo by a tic cell, since this
may also produce T
helper cell responses which are needed for an ive immune response (Ossendorp et al.,
Immunol Lett. (2000), 74:75-9; Ossendorp et a1. , J. Exp. Med. 3-702. In general, it
is possible to administer an effective amount of the tumor-associated antigen to a patient by
errnal injection, for example. r, injection may also be carried out intranodally into
a lymph node (Maloy et al. (2001), Proc Natl Acad Sci USA 983299-303).
According to the invention the term "artificial T cell receptor" is synonymous with the terms
ric T cell receptor" and "chimeric antigen receptor (CAR)".
These terms relate to engineered receptors, which confer an arbitrary specificity such as the
specificity of a monoclonal antibody onto an immune effector cell such as a T cell. In this way, a
large number of cancer—specific T cells can be generated for adoptive cell transfer. Thus, an
artificial T cell or ma" be present on T cells, e.g. instead of or in addition to the T cell's
own T cell receptor. Such T cells do not necessarily require processing and presentation of an
antigen for recognition of the target cell but rather may recognize preferably with specificity any
antigen present on a target cell. Preferably, said ial T cell receptor is expressed on the
surface of the cells. For the purpose of the present invention T cells comprising an artificial T
cell receptor are comprised by the term "T cell" as used herein.
In one embodiment, a single—chain variable fragment (scFv) derived from a monoclonal antibody
is fused to CD3-zeta transmembrane and endodomain. Such molecules result in the transmission
of a zeta signal in response to recognition by the scFv of its antigen target on a target cell and
killing of the target cell that expresses the target antigen. Antigen recognition domains which
also may be used include among others T-cell receptor (TCR) alpha and beta single chains. In
fact almost anything that binds a given target with high affinity can be used as an antigen
recognition domain.
Following antigen recognition, receptors cluster and a signal is itted to the cell. In this
respect, a "T cell signaling domain" is a domain, preferably an endodomain, which transmits an
activation signal to the T cell after antigen is bound. The most commonly used endodomain
ent is CD3 —zeta.
Adoptive cell transfer therapy with CAR-engineered T cells sing chimeric antigen
receptors is a promising anti-cancer therapeutic as dified T cells can be engineered to
target virtually any tumor antigen. For example, t‘s T cells may be genetically engineered
to express CARS specifically directed towards ns on the patient's tumor cells, then infused
back into the patient.
According to the invention an ial T cell receptor may replace the fimction of a T cell
receptor as described above and, in particular, may confer reactivity such as cytolytic activity to
a cell such as a T cell as described above. However, in contrast to the binding of the T cell
receptor to an antigen e-MHC complex as described above, an artificial T cell or
may bind to an antigen, in particular expressed on the cell surface.
The T—cell surface glycoprotein CD3-zeta chain is a protein that in humans is encoded by the
CD247 gene. CD3-zeta together with T-cell receptor alpha/beta and gamma/delta heterodimers
and CD3-gamma, -delta, and -epsilon, forms the T-cell or-CD3 complex. The zeta chain
plays an important role in coupling antigen recognition to several intracellular signal-
transduction pathways. The term "CD3-zeta" preferably relates to human CD3-zeta, and, in
particular, to a protein comprising, preferably consisting of the amino acid sequence of SEQ ID
NO: 45 of the sequence listing or a t of said amino acid sequence.
CD28 (Cluster of Differentiation 28) is one of the molecules expressed on T cells that provide
co-stimulatory signals, which are required for T cell activation. CD28 is the receptor for CD80
(B7.1) and CD86 (B72). Stimulation through CD28 in addition to the T cell receptor (TCR) can
provide a potent co~stimulatory signal to T cells for the production of various interleukins (IL-6
in particular). The term "CD28" preferably relates to human CD28, and, in particular, to a
protein comprising, ably consisting of the amino acid sequence of SEQ ID NO: 44 of the
sequence g or a variant of said amino acid sequence.
According to the invention, CARS may generally se three domains.
2015/056899
The first domain is the binding domain which recognizes and binds CLDN6.
The second domain is the mulation domain. The co—stimulation domain serves to enhance
the proliferation and survival of the cytotoxic lymphocytes upon binding of the CAR to a
targeted moiety. The identity of the co-stimulation domain is d only in that it has the ability
to enhance cellular proliferation and survival upon g of the targeted moiety by the CAR.
Suitable co-stimulation domains include CD28, CD137 (4-1813), a member of the tumor necrosis
factor (TNF) receptor family, CD134 (0X40), a member of the TNFR-superfamily of receptors,
and CD278 (ICOS), a uperfamily co-stimulatory molecule sed on activated T cells.
The skilled person will understand that sequence variants of these noted co-stimulation domains
can be used without adversely impacting the invention, where the variants have the same or
similar activity as the domain on which they are modeled. Such ts will have at least about
80% sequence identity to the amino acid sequence of the domain from which they are derived. In
some embodiments of the invention, the CAR constructs comprise two co-stimulation domains.
While the particular combinations include all possible variations of the four noted domains,
specific examples e CD28+CD137 (4-1 BB) and CD28+CD134 (0X40).
The third domain is the activation signaling domain (or T cell signaling domain). The activation
signaling domain serves to activate cytotoxic lymphocytes upon binding of the CAR to CLDN6.
The identity of the activation signaling domain is limited only in that it has the ability to induce
activation of the selected cytotoxic lymphocyte upon binding of the CLDN6 by the CAR.
Suitable tion signaling domains include the T cell CD3 [zeta] chain and Fe or
{gamma}. The skilled artisan will understand that sequence ts of these noted activation
signaling domains can be used without adversely ing the invention, where the variants
have the same or similar activity as the domain on which they are modeled. Such variants will
have at least about 80% sequence identity to the amino acid sequence of the domain from which
they are derived.
The CARS of the present invention may comprise the three domains, er in the form of a
fusion protein. Such fusion proteins will generally comprise a binding domain, one or more co-
stimulation domains, and an activation signaling domain, linked in a N-terminal to C-terminal
direction. However, the CARS of the present invention are not limited to this arrangement and
other arrangements are acceptable and include a binding domain, an activation signaling domain,
and one or more co-stimulation s. It will be understood that because the binding domain
must be free to bind CLDN6, the placement of the g domain in the fusion protein will
lly be such that display of the region on the exterior of the cell is achieved. In the same
, because the co-stimulation and activation signaling domains serve to induce activity and
proliferation of the cytotoxic lymphocytes, the fusion n will generally display these two
domains in the interior of the cell. The CARs may include additional elements, such as a signal
peptide to ensure proper export of the fusion protein to the cells surface, a transmembrane
domain to ensure the fusion protein is ined as an integral membrane protein, and a hinge
domain (or spacer region) that imparts flexibility to the binding domain and allows strong
binding to CLDN6.
The cells used in connection with the CAR system of the t invention are preferably T cells,
in particular xic lymphocytes, preferably selected from xic T cells, natural killer
(NK) cells, and lymphokine-activated killer (LAK) cells. Upon. activation, each of these
cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells
trigger the destruction of target cells by either or both of the following means. First, upon
tion T cells release cytotoxins such as in, granzymes, and ysin. Perforin and
granulysin create pores in the target cell, and granzymes enter the cell and trigger a caspase
cascade in the cytoplasm that induces apoptosis (programmed cell death) of the cell. Second,
apoptosis can be induced via Fas-Fas ligand interaction between the T cells and target tumor
cells. The cytotoxic cytes will preferably be autologous cells, although heterologous cells
or allogenic cells can be used.
According to the ion, a "reference" such as a reference sample or reference organism may
be used to correlate and compare the results obtained in the methods of the invention from a test
sample or test organism. Typically the reference organism is a healthy organism, in particular an
organism which does not suffer from a disease such as a cancer disease. A ence value" or
”reference level" can be determined from a reference empirically by measuring a sufficiently
large number of references. Preferably the reference value is determined by measuring at least 2,
preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably
at least 20, preferably at least 30, preferably at least 50, or preferably at least 100 references.
According to the invention, the term "binding agent" includes any compound that has a binding
capacity to a target. Preferably, such binding agent comprises at least one binding domain for the
target. The term includes molecules such as dies and dy fragments, bispecific or
multispecific molecules, chimeric antigen receptors (CARS) and all artificial binding molecules
olds) having a binding capacity to the target including but not limited to nanobodies,
affibodies, anticalins, DARPins, monobodies, avimers, and odies. In one ment
said binding is a specific binding.
The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to
antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are
characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic
globulin (1g) fold. The term encompasses membrane bound immunoglobulins as well as
soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface
immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble
immunoglobulins are generally termed antibodies. Immunoglobulins generally comprise several
chains, lly two identical heavy chains and two cal light chains which are linked via
disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the
V1, (variable light chain) domain, CL (constant light chain) domain, and the CH (constant heavy
chain) domains CH1, CH2, CH3, and CH4. There are five types of ian immunoglobulin
heavy chains, i.e., 0t, 8, a, y, and u which account for the different classes of dies, i.e., IgA,
IgD, IgE, IgG, and lgM. As d to the heavy chains of soluble globulins, the heavy
chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short
cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains,
i.e., lambda and kappa. The immunoglobulin chains comprise a le region and a constant
region. The constant region is essentially conserved within the different isotypes of the
globulins, wherein the variable part is highly divers and accounts for antigen
recognition.
The term “antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal
antibodies, recombinant antibodies, human antibodies, humanized antibodies and chimeric
antibodies. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein
as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable
region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can
be further subdivided into regions of hypervariability, termed complementarity determining
regions (CDR), interspersed with regions that are more ved, termed framework regions
(FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus
to carboxy-terminus in the ing order: FRl, CDRl, FRZ, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin
to host tissues or factors, including various cells of the immune system (e.g., effector cells) and
the first component (Clq) of the classical complement .
The term "monoclonal antibody" as used herein refers to a ation of antibody molecules of
single molecular composition. A monoclonal dy displays a single binding specificity and
affinity. In one embodiment, the onal antibodies are produced by a hybridoma which
includes a B cell obtained from a non-human animal, e.g., mouse, fused to an immortalized cell.
The term "recombinant antibody", as used herein, es all antibodies that are prepared,
expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an
animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the
immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host
cell transformed to express the antibody, e.g., from a transfectoma, (0) antibodies isolated from a
recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, d or
isolated by any other means that e splicing of immunoglobulin gene sequences to other
DNA sequences.
The term "human antibody", as used herein, is intended to include antibodies having variable and
constant regions derived from human germline immunoglobulin sequences. Human antibodies
may e amino acid residues not encoded by human ne immunoglobulin sequences
(e.g., mutations introduced by random or site—specific mutagenesis in vitro or by somatic
mutation in vivo).
The term "humanized antibody" refers to a molecule having an n binding site that is
substantially derived from an globulin from a non—human species, wherein the remaining
immunoglobulin structure of the molecule is based upon the structure and/or sequence of a
human immunoglobulin. The antigen binding site may either comprise complete variable
domains fused onto constant domains or only the mentarity determining s (CDR)
grafted onto appropriate framework s in the variable domains. Antigen binding sites may
be wild-type or modified by one or more amino acid substitutions, e.g. modified to resemble
human immunoglobulins more closely. Some forms of humanized dies preserve all CDR
sequences (for example a humanized mouse antibody which contains all six CDRs from the
mouse antibody). Other forms have one or more CDRs which are altered with respect to the
original antibody.
The term ”chimeric antibody" refers to those antibodies wherein one portion of each of the amino
acid sequences of heavy and light chains is homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a particular class, while the
remaining segment of the chain is homologous to corresponding sequences in another. Typically
the variable region of both light and heavy chains mimics the variable regions of antibodies
derived from one species of mammals, while the constant portions are homologous to sequences
of antibodies derived from another. One clear advantage to such chimeric forms is that the
variable region can conveniently be derived from tly known sources using readily
available B-cells or hybridomas from non-human host organisms in ation with constant
regions derived from, for example, human cell ations. While the variable region has the
age of ease of preparation and the city is not ed by the source, the constant
region being human, is less likely to elicit an immune response from a human subject when the
antibodies are ed than would the constant region from a non human source. However the
definition is not limited to this particular example.
Antibodies may be derived from different species, including but not limited to mouse, rat, rabbit,
guinea pig and human.
Antibodies described herein include IgA such as lgAl or IgAZ, lgGl, IgGZ, lgG3, lgG4, lgE,
IgM, and IgD dies. In various embodiments, the antibody is an IgGl antibody, more
ularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, K, 7t), an IgG2a antibody (e.g.
IgGZa, K, )t), an IgG2b antibody (e.g. IgG2b, K, it), an IgG3 antibody (e.g. IgG3, K, it) or an IgG4
antibody (e.g. IgG4, K, it).
The antibodies described herein are preferably isolated. An "isolated antibody" as used , is
intended to refer to an antibody which is substantially free of other antibodies having different
antigenic specificities (e.g., an isolated antibody that specifically binds to CLDN6 is
substantially free of antibodies that specifically bind antigens other than CLDN6). An isolated
antibody that specifically binds to an epitope, isoform or t of human CLDN6 may,
however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CLDN6
species homologs). er, an isolated antibody may be substantially free of other cellular
al and/or chemicals. In one embodiment of the invention, a combination of "isolated"
onal antibodies relates to antibodies having different specificrities and being combined in
a well defined composition or mixture.
The terms "antigen-binding portion" of an antibody (or simply "binding portion") or "antigen-
binding fragment" of an antibody (or simply "binding fragment") or similar terms refer to one or
more fragments of an antibody that retain the ability to specifically bind to an antigen. It has
been shown that the antigen—binding function of an dy can be performed by fragments of a
full—length dy. Examples of binding fragments encompassed within the term "antigen-
binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of
the VL, VH, CL and CH domains; (ii) F(ab')2 fragments, bivalent nts comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the
VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of
an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a
VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of
two or more isolated CDRs which may optionally be joined by a synthetic . Furthermore,
although the two domains of the Fv nt, VL and VH, are coded for by separate genes, they
can be joined, using recombinant methods, by a synthetic linker that enables them to be made as
a single protein chain in which the VL and VH regions pair to form monovalent molecules
(known as single chain Fv (scFV); see e.g., Bird et a1. (1988) Science 242: 423-426; and Huston
et a1. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also
intended to be encompassed within the term en-binding nt" of an antibody. A
further example is binding-domain immunoglobulin fusion ns comprising (i) a binding
domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an
immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an
immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The
binding domain polypeptide can be a heavy chain le region or a light chain variable region.
The binding-domain immunoglobulin fusion proteins are further disclosed in US 118592
and US 2003/0133939. These antibody fragments are obtained using conventional techniques
known to those with skill in the art, and the nts are screened for utility in the same manner
as are intact antibodies.
According to the invention, the term "binding domain for CLDN6" includes and preferably
relates to the antigen-binding portion of a CLDN6 antibody, i.e. an antibody which is directed
against CLDN6 and is ably specific for CLDN6.
The term "binding domain" characterizes in connection with the present invention a structure,
e.g. of an antibody, which binds to/interacts with a given target stmcture/antigen/epitope. Thus,
the binding domain according to the invention designates an "antigen-interaction~site".
All antibodies and derivatives of antibodies such as antibody nts as described herein for
the purposes of the invention are encompassed by the term ”antibody".
Antibodies can be ed by a y of techniques, including conventional monoclonal
antibody methodology, e.g., the rd somatic cell hybridization technique of Kohler and
Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred,
in principle, other ques for producing monoclonal dies can be employed, e.g., Viral
or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of
antibody genes.
The preferred animal system for preparing hybridomas that secrete onal antibodies is the
murine system. Hybridoma production in the mouse is a very well established procedure.
Immunization protocols and ques for isolation of immunized splenocytes for fusion are
known in the art. Fusion rs (e.g., murine myeloma cells) and fusion procedures are also
known.
Other preferred animal systems for preparing hybridomas that e monoclonal antibodies are
the rat and the rabbit system (e.g. described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A.
9229348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).
To generate dies, mice can be immunized with carrier-conjugated peptides derived from
the antigen sequence, i.e. the sequence against which the antibodies are to be directed, an
enriched preparation of recombinantly expressed antigen or fragments thereof and/or cells
expressing the antigen, as described. Alternatively, mice can be immunized with DNA encoding
the antigen or fragments thereof. In the event that immunizations using a purified or enriched
WO 50327
preparation of the antigen do not result in antibodies, mice can also be immunized with cells
expressing the n, e.g., a cell line, to promote immune responses.
The immune se can be monitored over the course of the immunization protocol with
plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient
titers of immunoglobulin can be used for fusions. Mice can be boosted intraperitonealy or
enously with antigen expressing cells 3 days before sacrifice and removal of the spleen to
increase the rate of specific antibody secreting hybridomas.
To generate hybridomas producing monoclonal antibodies, cytes and lymph node cells
from immunized mice can be isolated and fused to an appropriate alized cell line, such as
a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of
antigen-specific antibodies. dual wells can then be screened by ELISA for dy
secreting omas. By lmmunofluorescence and FACS analysis using antigen expressing
cells, antibodies with specificity for the antigen can be fied. The antibody secreting
hybridomas can be ed, screened again, and if still positive for monoclonal antibodies can be
ned by limiting dilution. The stable subclones can then be cultured in vitro to generate
antibody in tissue culture medium for characterization.
The ability of antibodies and other binding agents to bind an antigen can be determined using
standard binding assays (e.g., ELISA, Western Blot, lmmunofluorescence and flow cytometric
analysis).
Antibodies and derivatives of antibodies are useful for providing binding domains such as
antibody fragments, in particular for providing VL and VH regions.
A binding domain for CLDN6 which may be present within an artificial T cell receptor has the
ability of binding to CLDN6, i.e. the ability of g to an epitope present in CLDN6,
preferably an epitope located within the extracellular domains of CLDN6, in particular the first
extracellular loop, preferably amino acid positions 28 to 76 of CLDN6 or the second
extracellular loop, preferably amino acid positions 141 to 159 of CLDN6. In particular
embodiments, a binding domain for CLDN6 binds to an epitope on CLDN6 which is not present
on CLDN9. Preferably, a binding domain for CLDN6 binds to an epitope on CLDN6 which is
not present on CLDN4 and/or CLDN3. Most preferably, a binding domain for CLDN6 binds to
an epitope on CLDN6 which is not present on a CLDN protein other than CLDN6.
A g domain for CLDN6 preferably binds to CLDN6 but not to CLDN9 and preferably
does not bind to CLDN4 and/or CLDN3. Preferably, a binding domain for CLDN6 is specific for
CLDN6. Preferably, a binding domain for CLDN6 binds to CLDN6 expressed on the cell
surface. In particular preferred embodiments, a binding domain for CLDN6 binds to native
epitopes of CLDN6 present on the surface of living cells.
In a red embodiment, a binding domain for CLDN6 comprises a heavy chain variable
region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID
N05: 30, 32, 34 and 36 or a fragment thereof, or a variant of said amino acid sequence or
fragment.
In a preferred embodiment, a binding domain for CLDN6 comprises a light chain variable region
(VL) comprising an amino acid sequence selected from the group ting of SEQ ID NOs: 31,
33, 35, 37, 38 and 39 or a fragment f, or a variant of said amino acid sequence or fragment.
In certain preferred ments, a binding domain for CLDN6 comprises a combination of
heavy chain variable region (VH) and light chain variable region (VL) selected from the
following possibilities (i) to (xi):
(i) the VH comprises an amino acid sequence represented by SEQ ID NO: 30 or a fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 31 or a
fragment thereof,
(ii) the VH comprises an amino acid sequence represented by SEQ ID NO: 32 or a fragment
thereof and the VL comprises an amino acid sequence ented by SEQ ID NO: 33 or a
fragment thereof,
(iii) the VH comprises an amino acid sequence ented by SEQ ID NO: 34 or a fragment
thereof and the VL ses an amino acid sequence represented by SEQ ID NO: 35 or a
fragment f,
(iv) the VH comprises an amino acid ce represented by SEQ ID NO: 36 or a fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 37 or a
fragment thereof,
(v) the VH comprises an amino acid sequence represented by SEQ ID NO: 32 or a fragment
thereof and the VL ses an amino acid sequence represented by SEQ ID NO: 31 or a
fragment thereof,
(vi) the VH comprises an amino acid sequence represented by SEQ ID NO: 32 or a fragment
f and the VL comprises an amino acid sequence represented by SEQ ID NO: 38 or a
fragment thereof,
(vii) the VH comprises an amino acid sequence represented by SEQ ID NO: 32 or a fragment
thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 39 or a
fragment thereof.
In a particularly preferred ment, a binding domain for CLDN6 comprises the following
combination of heavy chain variable region (VH) and light chain variable region (VL):
the VH comprises an amino acid sequence ented by SEQ ID NO: 32 or a fragment thereof
and the VL comprises an amino acid sequence ented by SEQ ID NO: 39 or a nt
thereof.
The term "fragment" refers, in particular, to one or more of the complementarity-determining
regions (CDRs), preferably at least the CDR3 variable region, of the heavy chain variable region
(VH) and/or of the light chain variable region (VL). In one embodiment said one or more of the
complementarity-determining s (CDRs) are selected from a set of complementarity~
determining regions CDRl, CDRZ and CDR3. In a particularly preferred embodiment, the term
"fragment" refers to the complementarity—determining regions CDRl, CDRZ and CDR3 of the
heavy chain variable region (VH) and/or of the light chain variable region (VL).
In one embodiment a binding domain for CLDN6 comprising one or more CDRs, a set of CDRs
or a combination of sets of CDRs as described herein comprises said CDRs together with their
ening framework regions. Preferably, the portion will also include at least about 50% of
either or both of the first and fourth framework s, the 50% being the C-terminal 50% of the
first ork region and the N—terrninal 50% of the fourth framework region. Construction of
binding domains made by recombinant DNA ques may result in the introduction of
residues N— or C-terminal to the variable regions encoded by linkers introduced to tate
cloning or other manipulation steps, including the introduction of linkers to join variable regions
of the invention to further protein sequences ing immunoglobulin heavy chains, other
variable domains (for example in the production of diabodies) or protein labels.
In one embodiment a binding domain comprising one or more
CDRs, a set of CDRS or a
combination of sets of CDRs as described herein comprises said CDRs in a human antibody
framework.
The term "binding" according to the invention preferably relates to a specific binding.
According to the present invention, an agent such as a T cell receptor or an antibody is capable
of binding to a predetermined target if it has a significant affinity for said predetermined target
and binds to said predetermined target in standard assays. "Affinity" or "binding affinity" is often
measured by equilibrium dissociation constant (Kn). Preferably, the term ficant affinity"
refers to the binding to a predetermined target with a dissociation constant (K13) of 10‘5 M or
lower, 10'6 M or lower, 10‘7 M or lower, 10'8 M or lower, 10'9 M or lower, 10'10 M or lower, 10"1
M or lower, or 10'12 M or lower.
An agent is not (substantially) capable of binding to a target if it has no significant affinity for
said target and does not bind significantly, in particular does not bind detectably, to said target in
standard assays. Preferably, the agent does not detectably bind to said target if present in a
concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 ug/ml or
higher. ably, an agent has no cant affinity for a target if it binds to said target with a
KD that is at least Iii-fol , lOO—foid, 103-fold, 104-fold, lOs—fold, or IOG-fold higher than the K1)
for g to the predetermined target to which the agent is capable of binding. For e, if
the K1) for binding of an agent to the target to which the agent is capable of binding is 10'7 M, the
K0 for binding to a target for which the agent has no significant affinity would be at least 10'6 M,
-5 M, 10-4 M, 10-3 M, 10-2 M, or 10-1 M.
An agent is specific for a predetermined target if it is capable of binding to said predetermined
target while it is not antially) capable of g to other targets, i.e. has no significant
y for other targets and does not significantly bind to other targets in rd .
According to the invention, an agent is specific for CLDN6 if it is capable of binding to CLDN6
but is not (substantially) capable of g to other targets. Preferably, an agent is specific for
CLDN6 if the affinity for and the binding to such other targets does not cantly exceed the
affinity for or binding to CLDN6—unrelated proteins such as bovine serum albumin (BSA),
casein, human serum albumin (HSA) or non-claudin transmembrane proteins such as MHC
molecules or transferrin or or any other specified polypeptide. Preferably, an agent is
specific for a predetermined target if it binds to said target with a Kr) that is at least 10-fold, 100-
fold, 103-fold, 104-fold, 105-fold, or ld lower than the KB for binding to a target for which
it is not specific. For example, if the K13 for binding of an agent to the target for which it is
specific is 10'7 M, the KD for binding to a target for which it is not specific would be at least 10'6
M, 10-5 M, 10-4 M, 10-3 M, 10-2 M, or 10-1 M.
Binding of an agent to a target can be determined mentally using any suitable method; see,
for example, Berzofsky et al., ody-Antigen Interactions" In Fundamental Immunology,
Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman
and Company New York, N Y (1992), and s described herein. Affinities
may be readily
determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore
2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay
using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity
data may be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL,
51:660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if
measured under ent conditions, e.g., salt concentration, pH. Thus, measurements of affinity
and other antigen-binding ters, e.g., K13, ICso, are preferably made with standardized
solutions of antibody and n, and a standardized buffer.
It is to be understood that the peptide and protein agents described herein
may be provided in
vitro or in vivo in the form of a nucleic acid such as RNA encoding the agent and/or in the form
of a host cell comprising a c acid such as RNA encoding the agent. In ular, a variety
of methods may be used to introduce CAR constructs into T cells including non-viral-based
DNA transfection, transposon-based systems and viral—based systems. Non-Viral-based DNA
transfection has low risk of insertional mutagenesis. oson-based systems can integrate
transgenes more efficiently than plasmids that do not contain an integrating element. Viral-based
systems include the use of y—retroviruses and lentiviral vectors. y—Retroviruses are relatively easy
to produce, efficiently and permanently transduce T cells, and have preliminarily proven safe
from an integration standpoint in primary human T cells. Lentiviral vectors also efficiently and
permanently uce T cells but are more expensive to manufacture. They are also ially
safer than irus based systems.
The peptide and protein agents bed herein may be delivered to a patient by administering a
nucleic acid such as RNA encoding the agent and/or by administering a host cell comprising a
c acid such as RNA encoding the agent. A nucleic acid when administered to a patient
be present in naked form or in a suitable delivery vehicle such as in the form of mes or
viral particles, or within a host cell. The nucleic acid ed can e the agent over
extended time periods in a sustained manner mitigating the instability at least partially observed
for therapeutic proteins. If a nucleic acid is stered to a patient without being present
within a host cell, it is preferably taken up by cells of the patient for expression of the agent
encoded by the nucleic acid. If a c acid is administered to a patient while being present
within a host cell, it is preferably expressed by the host cell within the patient so as to produce
the agent encoded by the nucleic acid.
The term "nucleic acid", as used , is intended to include DNA and RNA such as genomic
DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic
acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT
RNA) or tic RNA. According to the invention, a nucleic acid is preferably an isolated
nucleic acid.
Nucleic acids may be comprised in a vector. The term "vector” as used herein includes any
vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors
such as lambda phage, viral vectors such as iral or baculoviral vectors, or artificial
chromosome vectors such as bacterial artificial somes (BAC), yeast artificial
chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as
well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and
generally contain a desired coding ce and appropriate DNA sequences necessary for the
expression of the operably linked coding sequence in a particular host organism (e.g., bacteria,
yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally
used to engineer and y a certain d DNA fragment and may lack functional
sequences
needed for expression of the desired DNA fragments.
In the context of the present invention, the term "RNA" relates to a molecule which comprises
ribonucleotide residues and preferably being entirely or substantially composed of cleotide
es. ucleotide" relates to a nucleotide with a hydroxyl
group at the 2’—position of a B-
D-ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated
RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly
WO 50327
produced RNA, as well as modified RNA that differs from naturally occurring RNA by the
addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can
e on of non-nucleotide material, such as to the end(s) of a RNA or internally, for
example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also
comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically
synthesized nucleotides or ucleotides. These d RNAs can be referred to as analogs
or analogs of naturally-occurring RNA.
According to the present invention, the term "RNA" includes and preferably relates to ”mRNA"
which means "messenger RNA" and relates to a "transcript" which
may be ed using DNA
as template and encodes a peptide or protein. mRNA typically ses a 5‘ non translated
region (5'-UTR), a protein or peptide coding region and a 3' non translated region R).
mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro
transcription using a DNA template. In one embodiment of the invention, the RNA is obtained
by in vitro transcription or chemical synthesis. The in vitro transcription ology is known
to the skilled person. For example, there is a variety of in vitro transcription kits cially
available.
In one embodiment of the present invention, RNA is self-replicating RNA, such as single
stranded self-replicating RNA. In one embodiment, the self—replicating RNA is single stranded
RNA of positive sense. In one ment, the self-replicating RNA is viral RNA or RNA
derived from viral RNA. In one embodiment, the self-replicating RNA is alphaviral genomic
RNA or is derived from alphaviral genomic RNA. In one embodiment, the eplicating RNA
is a viral gene expression vector. In one embodiment, the virus is Semliki forest Virus. In one
embodiment, the eplicating RNA contains one or more transgenes at least one of said
transgenes encoding the agents described herein. In one embodiment, if the RNA is viral RNA or
derived from viral RNA, the transgenes may partially or completely replace viral
sequences such
as viral sequences encoding structural proteins. In one ment, the self-replicating RNA is
in vitro transcribed RNA.
In order to increase expression and/or stability of the RNA used according to the present
invention, it may be modified, preferably without altering the sequence of the expressed peptide
or protein.
The term "modification" in the context of RNA as used according to the present invention
includes any modification of RNA which is not naturally present in said RNA.
In one embodiment of the ion, the RNA used ing to the invention does not have
uncapped 5'—triphosphates. Removal of such uncapped 5'—triphosphates can be achieved by
treating RNA with a phosphatase.
The RNA according to the invention may have modified naturally occurring or synthetic
ribonucleotides in order to increase its stability and/or decrease cytotoxicity. For example, in one
embodiment, in the RNA used according to the invention 5—methylcytidine is substituted
partially or completely, preferably completely, for cytidine. Alternatively or additionally, in one
embodiment, in the RNA used according to the invention uridine is substituted partially or
completely, preferably tely, for uridine.
In one embodiment, the term cation" relates to providing an RNA with a 5’-cap or 5’-cap
analog. The term "5’-cap" refers to a cap structure found on the S'-end of an mRNA molecule
and generally consists of a guanosine nucleotide connected to the mRNA Via an unusual 5' to 5'
triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. The
term "conventional 5’-cap" refers to a naturally occurring RNA 5’-cap, preferably to the 7-
methylguanosine cap (m7G). In the context of the present invention, the term "5’—cap" includes a
’—cap analog that resembles the RNA cap ure and is modified to possess the ability to
stabilize RNA if ed thereto, preferably in vivo and/or in a cell.
Providing an RNA with a 5’-cap or 5’-cap analog may be achieved by in Vitro transcription of a
DNA te in the presence of said 5’—cap or 5’-cap analog, wherein said 5’-cap is co-
transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for
example, by in Vitro transcription, and the 5’-cap may be attached to the RNA post-
transcriptionally using capping enzymes, for example, capping enzymes of ia Virus.
The RNA may se further modifications. For example, a r modification of the RNA
used in the present invention may be an extension or truncation of the naturally occurring
poly(A) tail or an alteration of the 5'- or 3'-untranslated regions (UTR) such as uction of a
UTR which is not d to the coding region of said RNA, for example, the insertion of one or
more, preferably two copies of a 3'—UTR derived from a globin gene, such as alphaZ-globin,
alphal-globin, beta-globin, ably beta—globin, more preferably human beta—globin.
Therefore, in order to increase stability and/or expression of the RNA used according to the
present invention, it may be modified so as to be t in conjunction with a poly-A sequence,
preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to
200 and especially 100 to 150 adenosine residues. In an especially preferred embodiment the
poly—A sequence has a length of approximately 120 adenosine residues. In addition,
incorporation of two or more 3'—non translated regions (UTR) into the 3'-non translated region of
an RNA molecule can result in an enhancement in translation efficiency. In one particular
embodiment the 3’-UTR is derived from the human B-globin gene.
The term "stability" of RNA s to the "half-life" of RNA. life" relates to the period of
time which is needed to eliminate half of the activity, amount, or number of molecules. In the
context of the present invention, the half~life of an RNA is indicative for the stability of said
RNA. The ife of RNA may influence the "duration of expression" of the RNA. It can be
expected that RNA having a long half-life will be expressed for an extended time period.
In the context of the present invention, the term "transcription“ relates to a s, wherein the
genetic code in a DNA sequence is transcribed into RNA. uently, the RNA may be
ated into protein. According to the t invention, the term ”transcription” comprises "in
vitro transcription”, wherein the term "in vitro transcription“ relates to a process wherein RNA,
in particular mRNA, is in vitro synthesized in a cell-free system, preferably using appropriate
cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These
cloning vectors are generally designated as transcription vectors and are according to the t
invention encompassed by the term "vector”.
The term "translation" according to the invention s to the process in the ribosomes of a cell
by which a strand of ger RNA directs the assembly of a sequence of amino acids to make
a peptide or n.
Nucleic acids may, according to the ion, be present alone or in combination with other
nucleic acids, which may be homologous or heterologous. In preferred embodiments, a nucleic
acid is functionally linked to expression control sequences which may be homologous or
logous with respect to said nucleic acid. The term "homologous" means that the nucleic
2015/056899
acids are also functionally linked naturally and the term "heterologous" means that the nucleic
acids are not functionally linked lly.
A nucleic acid and an expression control sequence are "functionally" linked to one another, if
they are covalently linked to one another in such a way that expression or transcription of said
nucleic acid is under the control or under the influence of said expression control
sequence. If the
c acid is to be ated into a functional n, then, with an expression control
sequence functionally linked to a coding sequence, induction of said expression control sequence
results in transcription of said nucleic acid, without g a frame shift in the coding
sequence
or said coding sequence not being capable of being translated into the desired protein
or peptide.
The term ”expression control sequence" or "expression l element" comprises according to
the invention promoters, ribosome binding sites, enhancers and other control elements which
regulate transcription of a gene or translation of a mRNA. In particular embodiments of the
invention, the expression control sequences can be regulated. The exact structure of expression
control sequences may vary as a function of the species or cell type, but generally comprises 5’-
untranscribed and 5’- and 3’-untranslated sequences which are involved in initiation of
transcription and translation, respectively, such as TATA box, g sequence, CAAT
sequence, and the like. More cally, 5’—untranscribed expression control sequences
se a promoter region which includes a promoter sequence for transcriptional control of the
functionally linked c acid. sion control sequences may also comprise enhancer
sequences or upstream tor sequences.
The term "expression" is used according to the invention in its most general meaning and
comprises the production of RNA and/or peptides or proteins, e.g. by transcription and/or
translation. With respect to RNA, the term "expression" or "translation" relates in ular to
the production of peptides or ns. It also comprises partial expression of nucleic acids.
Moreover, expression can be transient or stable. According to the invention, the term expression
also includes an ”aberrant expression" or "abnormal expression".
"Aberrant expression" or "abnormal expression" means according to the invention that
expression is altered, preferably increased, compared to a reference, e.g. a state in a subject not
having a disease associated with aberrant or abnormal sion of a certain protein, e.g., a
tumor antigen. An increase in expression refers to an se by at least 10%, in particular at
least 20%, at least 50% or at least 100%, or more. In one embodiment, expression is only found
in a diseased tissue, while expression in a healthy tissue is repressed.
The term "specifically expressed" means that a protein is essentially only expressed in a specific
tissue or organ. For example, a tumor antigen specifically sed in gastric mucosa means
that said protein is primarily expressed in gastric mucosa and is not expressed in other tissues or
is not expressed to a significant extent in other tissue or organ types. Thus, a protein that is
exclusively expressed in cells of the gastric mucosa and to a significantly lesser extent in any
other tissue, such as testis, is specifically expressed in cells of the gastric mucosa. In some
embodiments, a tumor antigen may also be specifically expressed under normal conditions in
more than one tissue type or organ, such as in 2 or 3 tissue types or organs, but preferably in not
more than 3 ent tissue or organ types. In this case, the tumor antigen is then specifically
expressed in these organs. For example, if a tumor antigen is expressed under normal conditions
ably to an approximately equal extent in lung and stomach, said tumor antigen is
specifically expressed in lung and stomach.
According to the invention, the term "nucleic acid ng" means that c acid, if present
in the appropriate environment, preferably within a cell, can be expressed to produce a protein or
peptide it encodes.
Some aspects of the ion rely on the adoptive transfer of host cells which are transfected in
vitro with a nucleic acid such as RNA encoding an agent described herein and transferred to
recipients such as patients, preferably after ex vivo expansion from low precursor frequencies to
clinically relevant cell numbers. The host cells used for treatment according to the invention may
be autologous, allogeneic, or eic to a treated recipient.
The term "autologous" is used to describe anything that is derived from the same subject. For
example, "autologous transplan " refers to a transplant of tissue or organs derived from the same
subject. Such procedures are advantageous because they overcome the immunological r
which ise results in ion.
The term "allogeneic" is used to describe anything that is d from different individuals of
the same species. Two or more individuals are said to be neic to one another when the
genes at one or more loci are not identical.
The term "syngeneic" is used to describe anything that is derived from individuals or tissues
having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their
tissues.
The term "heterologous" is used to describe something consisting of multiple different elements.
As an example, the transfer of one individual's bone marrow into a different individual
constitutes a logous transplant. A heterologous gene is a gene derived from a source other
than the subject.
The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell.
For purposes of the present invention, the term fection" also includes the introduction of a
c acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be
present in a subject, e.g., a patient. Thus, according to the present invention, a cell for
transfection of a nucleic acid described herein can be present in vitro or in viva, e.
g. the cell can
form part of an organ, a tissue and/or an organism of a patient. According to the invention,
transfection can be transient or stable. For some applications of transfection, it is sufficient if the
transfected genetic al is only transiently expressed. Since the nucleic acid introduced in the
ection process is usually not ated into the nuclear genome, the n nucleic acid
will be d through s or degraded. Cells allowing epison'ial amplification of nucleic
acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually
remains in the genome of the cell and its daughter cells, a stable transfection must occur. RNA
can be transfected into cells to transiently express its coded protein.
According to the present invention, any technique useful for introducing, i.e. transferring or
transfecting, nucleic acids into cells may be used. Preferably, RNA is ected into cells by
standard techniques. Such techniques include electroporation, lipofection and microinjection. In
one ularly preferred ment of the present ion, RNA is introduced into cells by
electroporation.
Electroporation or electropermeabilization relates to a significant increase in the electrical
conductivity and permeability of the cell plasma membrane caused by an externally applied
electrical field. It is usually used in molecular biology as a way of introducing some substance
into a cell.
According to the invention it is preferred that introduction of nucleic acid encoding a protein or
peptide into cells results in expression of said protein or e.
The term "peptide" according to the invention ses oligo- and polypeptides and refers to
substances sing two or more, preferably 3 or more, preferably 4 or
more, preferably 6 or
more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more,
ably 16 more, ably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in
ular 100 amino acids joined covalently by peptide bonds. The term "protein" refers to large
peptides, ably to peptides with more than 100 amino acid residues, but in general the terms
"peptides" and "proteins" are synonyms and are used interchangeably herein.
According to the invention, a peptide may include natural amino acids and non-natural amino
acids. In one embodiment, a peptide merely includes natural amino acids.
According to the invention, the term "non-natural amino acid" refers to an amino acid having a
structure different from those of the 20 natural amino acid species. Since non-natural amino
acids have structures similar to those of natural amino acids, non-natural amino acids may be
classified as derivatives or s of given natural amino acids.
Preferably, the proteins and peptides described according to the invention have been isolated.
The terms ted protein" or "isolated peptide" mean that the protein or peptide has been
separated from its natural environment. An ed protein or peptide may be in an essentially
purified state. The term "essentially purified" means that the protein or peptide is essentially free
of other substances with which it is associated in nature or in vivo.
The teaching given herein with respect to specific amino acid
sequences, e.g. those shown in the
sequence listing, is to be construed so as to also relate to variants of said c sequences
resulting in sequences which are functionally equivalent to said specific sequences, e.g. amino
acid sequences exhibiting properties identical or r to those of the specific amino acid
sequences. One important property is to retain g of a peptide to an MHC molecule and/or
to a T cell receptor or of a T cell receptor to its target or to sustain effector functions of
a T cell.
Preferably, a sequence modified with respect to a specific sequence, when it replaces the specific
sequence in a T cell receptor s g of said T cell or to the target and ably
ons of said T cell receptor or T cell carrying the T cell receptor as described herein.
For example, the sequences shown in the sequence listing can be modified so as to remove one
or more, preferably all free cysteine residues, in particular by replacing the cysteine residues by
amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, leucine
or methionine, most preferably alanine or serine. For example, the cysteine at position 45 of the
ce shown in SEQ ID NO: 33 of the sequence listing or the corresponding cysteine in a
ce comprising said sequence may be modified in this way.
It will be appreciated by those skilled in the art that in particular the sequences of the CDR
sequences, hypervariable and variable regions can be modified without losing the ability to bind
to a target. For e, CDR regions will be either identical or highly homologous to the
regions of antibodies specified herein. By "highly homologous" it is contemplated that from 1 to
, ably from 1 to 4, such as l to 3 or I or 2 substitutions may be made in the CDRs. In
addition, the hypervariable and variable regions may be modified so that they show substantial
gy with the regions specifically disclosed herein.
A peptide ”variant" may retain the immunogenicity of a given peptide (e.g. the ability of the
variant to react with T cell lines or clones is not substantially diminished relative to the given
peptide). In other words, the abiiity of a variant to react with T cell lines or clones may be
enhanced or unchanged, relative to the given e, or may be diminished by less than 50%,
and preferably less than 20%, relative to the given peptide.
A variant may be identified by evaluating its ability to bind to a MHC molecule. In one preferred
embodiment, a variant peptide has a modification such that the ability of the variant peptide to
bind to a MHC le is increased relative to the given peptide. The ability of the variant
peptide to bind to a MHC molecule may be increased by at least 2-fold, preferably at least 3-fold,
4-fold, or 5—fold relative to that of a given peptide. Accordingly, within certain preferred
embodiments, a e comprises a variant in which I to 3 amino acid resides within an
immunogenic portion are substituted such that the y to react with T cell lines or clones is
tically r than that for the unmodified peptide. Such substitutions are preferably
located within an MHC binding site of the peptide. Preferred substitutions allow increased
binding to MHC class I or class II molecules. Certain variants contain conservative substitutions.
The term "variant" according to the invention also includes mutants, splice variants,
conformations, isoforms, allelic variants, species variants and species gs, in particular
those which are naturally present. An allelic variant relates to an alteration in the normal
sequence of a gene, the significance of which is often unclear. te gene sequencing often
identifies numerous allelic variants for a given gene. A species homolog is a nucleic acid or
amino acid sequence with a different species of origin from that of a given nucleic acid or amino
acid sequence. The term "varian " shall encompass any posttranslationally modified variants and
conformation variants.
For the es of the t invention, "variants" of an amino acid
sequence comprise amino
acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino
acid substitution variants. Amino acid deletion variants that comprise the deletion at the N-
terminal and/or C-terminal end of the n are also called N—terminal and/or C-terminal
truncation variants.
Amino acid insertion variants comprise ions of single or two or more amino acids in a
particular amino acid sequence. In the case of amino acid ce variants having an insertion,
one or more amino acid residues are inserted into a particular site in an amino acid ce,
although random insertion with appropriate screening of the resulting product is also possible.
Amino acid addition ts comprise amino- and/or carboxy-tenninal fusions of one or more
amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
Amino acid deletion variants are characterized by the removal of one or more amino acids from
the ce, such as by removal of l, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions
may be in any position of the protein.
Amino acid tution variants are characterized by at least one residue in the sequence being
removed and another residue being inserted in its place. Preference is given to the modifications
being in positions in the amino acid sequence which are not conserved between homologous
proteins or peptides and/or to replacing amino acids with other ones having similar properties.
Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e.,
tutions of similarly charged or uncharged amino acids. A conservative amino acid change
involves substitution of one of a family of amino acids which are related in their side chains.
Naturally occurring amino acids are generally divided into four families: acidic (aspartate,
glutamate), basic (lysine, arginine, histidine), non—polar (alanine, valine, leucine, isoleucine,
e, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine,
glutarnine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and
tyrosine are sometimes classified jointly as aromatic amino acids.
Preferably the degree of similarity, preferably ty between a given amino acid sequence and
an amino acid sequence which is a variant of said given amino acid sequence will be at least
about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is
given preferably for an amino acid region which is at least about 10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%,
at least about 80%, at least about 90% or about 100% of the entire length of the reference amino
acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids,
the degree of similarity or identity is given preferably for at least about 20, at least about 40, at
least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least
about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. In
preferred ments, the degree of similarity or ty is given for the entire length of the
reference amino acid sequence. The alignment for ining
ce similarity, preferably
sequence identity can be done with art known tools, preferably using the best sequence
alignment, for example, using Align, using standard settings, preferably EMBOSS::needle,
Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
"Sequence similarity" indicates the percentage of amino acids that either are cal or that
represent conservative amino acid substitutions. "Sequence identity" between two amino acid
sequences indicates the percentage of amino acids that are identical between the ces.
The term ntage identity" is intended to denote a percentage of amino acid residues which
are identical n the two sequences to be compared, obtained after the best alignment, this
tage being purely statistical and the differences between the two ces being
distributed randomly and over their entire length. Sequence comparisons between two amino
acid sequences are conventionally carried out by comparing these
sequences after having aligned
them optimally, said comparison being carried out by segment or by "window of comparison" in
order to identify and compare local regions of sequence similarity. The optimal alignment of the
sequences for comparison may be produced, besides manually, by means of the local homology
algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local
homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the
similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or
by means of computer ms which use these algorithms (GAP, BESTFIT, FASTA, BLAST
P, BLAST N and TFASTA in Wisconsin Genetics Software Package. Genetics Computer Group,
575 Science Drive, Madison, Wis).
The percentage ty is calculated by determining the number of identical positions between
the two sequences being compared, ng this number by the number of ons compared
and multiplying the result obtained by 100 so as to obtain the percentage identity n these
two sequences.
Homologous amino acid sequences exhibit according to the invention at least 40%, in ular
at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at
least 98 or at least 99% identity of the amino acid residues.
The amino acid ce ts bed herein may readily be prepared by the skilled
person, for example, by recombinant DNA lation. The manipulation of DNA sequences
for ing proteins and peptides having substitutions, additions, insertions or deletions, is
described in detail in Sambrook ct al. (1989), for example. Furthermore, the peptides and amino
acid variants described herein may be readily prepared with the aid of known peptide synthesis
techniques such as, for example, by solid phase sis and similar methods.
The invention includes derivatives of the peptides or proteins described herein which are
comprised by the terms "peptide" and ”protein". According to the invention, "derivatives" of
proteins and peptides are modified forms of proteins and es. Such modifications include
any chemical modification and comprise single or multiple substitutions, deletions and/or
additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids
and/or proteins or peptides. In one embodiment, ”derivatives" of proteins or peptides include
those modified analogs resulting from glycosylation, acetylation, phosphorylation, amidation,
palmitoylation, myristoylation, isoprenylation, tion, alkylation, derivatization, introduction
of protective/blocking groups, proteolytic cleavage or g to an antibody or to another
cellular ligand. The term "derivative" also extends to all functional chemical equivalents of said
proteins and peptides. Preferably, a modified peptide has increased stability and/or increased
immunogenicity.
Also included are mimetics of peptides. Such mimetics may comprise amino acids linked to one
or more amino acid mimetics (i e., one or more amino acids within the peptide may be replaced
by an amino acid mimetic) or may be entirely nonpeptide mimetics. An amino acid mimetic is a
compound that is conformationally similar to an amino acid, e. g. such that it can be substituted
for an amino acid without substantially diminishing the ability to react with T cell lines or
clones. A nonpeptide c is a compound that does not contain amino acids, and that has an
overall conformation that is similar to a peptide, eg. such that the ability of the mimetic to react
with T cell lines or clones is not substantially diminished relative to the ability of a given
peptide.
According to the ion, a variant, tive, d form, fragment, part or portion of an
amino acid sequence, peptide or protein preferably has a functional property of the amino acid
sequence, peptide or protein, respectively, from which it has been derived, i.e. it is functionally
equivalent. In one embodiment, a variant, derivative, d form, fragment, part or portion of
an amino acid sequence, peptide or protein is immunologically equivalent to the amino acid
sequence, peptide or protein, respectively, from which it has been derived. In one embodiment,
the fimetional property is an immunological property.
A particular property is the ability to form a complex with MHC molecules and, where
riate, generate an immune response, preferably by stimulating cytotoxic or T helper cells.
The term "immunologically lent" means that the immunologically equivalent molecule
such as the immunologically equivalent amino acid sequence exhibits the same or ially the
same immunological properties and/or exerts the same or essentially the same logical
effects, e.g., with t to the type of the immunological effect such as induction of a humeral
and/or cellular immune response, the th and/or duration of the induced immune reaction, or
the specificity of the induced immune reaction. In the t of the present invention, the term
ologically equivalent" is preferably used with t to the immunological effects or
properties of a peptide or e variant used for immunization. For example, an amino acid
sequence is immunologically equivalent to a reference amino acid sequence if said amino acid
sequence when exposed to the immune system of a subject induces an immune reaction having a
specificity of ng with the reference amino acid sequence.
The term ”derived" means according to the invention that a particular entity, in particular a
particular sequence, is present in the object from which it is derived, in particular an organism or
molecule. In the case of amino acid sequences, especially ular
sequence regions, "derived"
in particular means that the relevant amino acid sequence is derived from an amino acid
sequence in which it is present.
The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell with an intact
membrane that has not released its normal intracellular components such as
enzymes, organelles,
or genetic material. An intact cell preferably is a Viable cell, i.e. a living cell capable of carrying
out its normal metabolic functions. Preferably said term relates according to the ion to
cell which can be transfected with an ous nucleic acid. Preferably, the cell when
transfected with an exogenous nucleic acid and transferred to a ent can
express the nucleic
acid in the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells,
fungal cells or mammalian cells. Suitable bacterial cells include cells from gram-negative
bacterial strains such as strains of ichia coli, Proteus, and Pseudomonas, and gram—
positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and
Lactococcus. Suitable fungal cell include cells from species of Trichoderma, Neurospora, and
illus. Suitable yeast cells include cells from species of Saccharomyces (Tor example
Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizo saccharomyces pombe),
Pichia (for example Pichia pastoris and Pichia methanolicd), and Hansenula. Suitable
mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK
and the like. However, amphibian cells, insect cells, plant cells, and
any other cells used in the
art for the expression of heterologous proteins can be used as well. ian cells are
particularly preferred for ve transfer, such as cells from humans, mice, hamsters, pigs,
goats, and primates. The cells may be derived from a large number of tissue types and include
primary cells and cell lines such as cells of the immune , in particular antigen-presenting
cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and
hymal stem cells and other cell types. An antigen-presenting cell is a cell that displays
antigen in the context of major ompatibility complex on its surface. T cells may recognize
this complex using their T cell receptor (TCR).
A cell which comprises a nucleic acid molecule preferably s the e or n
encoded by the nucleic acid.
The cell may be a recombinant cell and may secrete the encoded peptide or protein,
may express
it on the surface and preferably may additionally s an MHC molecule which binds to said
peptide or protein or a procession product thereof. In one embodiment, the cell expresses the
MHC molecule endogenously. In a further embodiment, the cell
expresses the MHC molecule
and/or the peptide or protein or the procession product thereof in a recombinant manner. The cell
is preferably liferative. In a preferred embodiment, the cell is an antigen-presenting cell,
in particular a dendritic cell, a monocyte or a macrophage.
The term "clonal ion" refers to a process wherein a specific entity is multiplied. In the
context of the present invention, the term is preferably used in the context of an immunological
response in which lymphocytes are stimulated by an antigen, proliferate, and the specific
lymphocyte recognizing said antigen is amplified. Preferably, clonal expansion leads to
differentiation of the lymphocytes.
A disease associated with n expression may be detected based on the
presence of T cells
that specifically react with a peptide in a biological sample. Within certain methods, a biological
sample comprising CD4+ and/or CD8+ T cells isolated from a t is incubated with a peptide
of the invention, a nucleic acid encoding such peptide and/or an antigen-presenting cell that
expresses and/or presents at least an immunogenic portion of such a peptide, and the presence or
absence of specific activation of the T cells is detected. Suitable biological samples include, but
are not d to, isolated T cells. For e, T cells may be isolated from a patient by e
ques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood
lymphocytes). For CD4+ T cells, activation is preferably detected by evaluating proliferation of
the T cells. For CD8+ T cells, activation is preferably detected by evaluating cytolytic activity. A
level of proliferation that is at least two fold r and/or a level of tic activity that is at
least 20% greater than in disease-free subjects indicates the presence of a disease ated with
antigen expression in the subject.
e" or "inhibit" as used herein means the ability to cause an overall decrease, preferably of
% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most
preferably of 75% or r, in the level. The term "inhibit" or similar phrases includes a
complete or essentially complete inhibition, i.e. a reduction to zero or ially to zero.
Terms such as "increase" or "enhance" preferably relate to an increase or enhancement by about
at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%,
more preferably at least 50%, even more preferably at least 80%, and most preferably at least
100%.
The agents, compositions and methods bed herein can be used to treat a subject with a
disease, e.g., a disease characterized by the presence of diseased cells expressing CLDN6 and
preferably ting CLDN6 in the context of MHC molecules. Examples of diseases which
can be treated and/or prevented encompass all diseases expressing CLDN6. Particularly
preferred es are cancer diseases.
The agents, compositions and methods described herein
may also be used for immunization or
vaccination to prevent a disease described herein.
The terms "normal tissue“ or "normal conditions" refer to healthy tissue or the conditions in
healthy subject, i.e., non-pathological conditions, wherein ”healthy" preferably means noncancerous
The term "disease" refers to an abnormal condition that affects the body of an individual. A
disease is often construed as a medical condition associated with c symptoms and signs. A
disease may be caused by factors originally from an external , such as infectious disease,
or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease"
is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social
problems, or death to the individual afflicted, or similar ms for those in contact with the
individual. In this broader sense, it sometimes includes injuries, disabilities, disorders,
syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure
and function, while in other contexts and for other purposes these may be considered
distinguishable categories. Diseases y affect individuals not only physically, but also
emotionally, as cting and living with many diseases can alter one's perspective on life, and
one's ality. According to the ion, the term se“ es
cancer, in particular
those forms of cancer described . Any reference herein to cancer or particular forms of
cancer also includes cancer metastasis thereof. In a preferred embodiment, a disease to be treated
according to the present application involves cells expressing CLDN6 and optionally presenting
CLDN6 in the context ofMHC les.
ses involving cells expressing CLDN6" or similar expressions means according to the
invention that CLDN6 is expressed in cells of a diseased tissue or
organ. In one embodiment,
sion of CLDN6 in cells of a ed tissue or organ is increased compared to the state in a
healthy tissue or organ. An increase refers to an increase by at least 10%, in ular at least
%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%
or even more. In one embodiment, expression is only found in a diseased tissue, while expression
in a healthy tissue is repressed. According to the invention, diseases involving cells expressing
CLDN6 include cancer diseases. Furthermore, according to the invention, cancer diseases
preferably are those wherein the cancer cells express CLDN6.
The terms "cancer disease" or "cancer" refer to or describe the physiological condition in an
individual that is typically terized by unregulated cell growth. Examples of cancers
include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
ularly, examples of such cancers include bone cancer, blood , lung cancer, liver
cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular
melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, colon cancer, breast cancer, te cancer, uterine , carcinoma of the sexual and
uctive , Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine,
cancer of the endocrine , cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, a of soft tissue, cancer of the bladder, cancer of the kidney,
renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system
(CNS), neuroectoderrnal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.
The term r" according to the invention also comprises cancer metastases. Preferably, a
"cancer disease" is characterized by cells expressing CLDN6 and a cancer cell expresses
CLDN6.
A diseased cell ably is a cell expressing CLDN6 said CLDN6 preferably being present on
the surface of said cell as transmembrane protein and/or being presented by said cell in the
context ofMHC such as MHC I. A cell expressing CLDN6 preferably is a cancer cell, preferably
of the cancers described herein.
2015/056899
In one embodiment, a cancer disease is a malignant disease which is characterized by the
properties of anaplasia, veness, and metastasis. A malignant tumor may be contrasted with
a non-cancerous benign tumor in that a malignancy is not self-limited in its , is capable of
invading into adjacent tissues, and may be capable of spreading to distant tissues (metastasizing),
while a benign tumor has none of those properties.
According to the invention, the term "tumor" or "tumor disease" refers to a swelling or lesion
formed by an abnormal growth of cells (called neoplastic cells or tumor cells). By "tumor cell" is
meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and ues to
grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack
of structural organization and functional coordination with the nomal tissue, and y form
distinct mass of tissue, which may be either benign, lignant or malignant.
According to the ion, a "carcinoma" is a malignant tumor derived from epithelial cells.
This group represents the most common cancers, including the common forms of breast,
prostate, lung and colon .
"Adenocarcinoma" is a cancer that originates in glandular tissue. This tissue is also part of a
larger tissue category known as epithelial tissue. lial tissue includes skin, glands and a
variety of other tissue that lines the cavities and organs of the body. lium is derived
embryologically from ectoderm, endoderrn and mesoderm. To be classified as adenocarcinoma,
the cells do not necessarily need to be part of a gland, as long as they have secretory ties.
This form of carcinoma can occur in some higher mammals, including humans. Well
differentiated adenocarcinomas tend to resemble the glandular tissue that they are derived from,
while poorly differentiated may not. By staining the cells from a biopsy, a pathologist will
determine whether the tumor is an adenocarcinoma or some other type of cancer.
Adenocarcinomas can arise in many tissues of the body due to the ubiquitous nature of glands
within the body. While each gland may not be secreting the same substance, as long
as there is
an exocrine function to the cell, it is considered glandular and its ant form is therefore
named adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize
given enough time to do so. Ovarian arcinoma is the most common type of ovarian
carcinoma. It includes the serous and mucinous adenocarcinomas, the clear cell adenocarcinoma
and the endometrioid adenocarcinoma.
Lymphoma and leukemia are malignancies derived from hematopoietic -forming) cells.
Blastic tumor or blastoma is a tumor (usually malignant) which resembles an immature or
embryonic tissue. Many of these tumors are most common in children.
By ”metastasis” is meant the spread of cancer cells from its original site to another part of the
body. The formation of metastasis is a very complex process and depends on detachment of
ant cells from the y tumor, on of the extracellular matrix, penetration of the
endothelial basement membranes to enter the body cavity and vessels, and then, after being
transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the
target site s on angiogenesis. Tumor metastasis often occurs even after the removal of the
primary tumor because tumor cells or components may remain and develop metastatic potential.
In one embodiment, the term "metastasis" according to the invention relates to "distant
metastasis" which relates to a asis which is remote from the primary tumor and the
regional lymph node system. In one embodiment, the term "metastasis" according to the
invention s to lymph node metastasis.
The cells of a secondary or metastatic tumor are like those in the original tumor. This means, for
example, that, if ovarian cancer metastasizes to the liver, the secondary tumor is made up of
abnormal ovarian cells, not of abnormal liver cells. The tumor in the liver is then called
metastatic ovarian cancer, not liver cancer.
A relapse or recurrence occurs when a person is affected again by a condition that affected them
in the past. For example, if a patient has suffered from a tumor disease, has received a successful
treatment of said disease and again develops said disease said newly developed disease may be
considered as relapse or ence. However, according to the invention, a relapse or ence
of a tumor disease may but does not necessarily occur at the site of the original tumor e.
Thus, for example, if a patient has ed from ovarian tumor and has received a successful
treatment a relapse or recurrence may be the occurrence of an ovarian tumor or the occurrence of
a tumor at a site different to ovary. A relapse or recurrence of a tumor also includes situations
wherein a tumor occurs at a site ent to the site of the original tumor as well as at the site of
the original tumor. ably, the original tumor for which the patient has received a treatment
is a primary tumor and the tumor at a site different to the site of the original tumor is a secondary
or metastatic tumor.
The term ”treatment" or "therapeutic treatment" relates to any treatment which improves the
health status and/or prolongs (increases) the lifespan of an individual. Said treatment may
eliminate the e in an individual, arrest or slow the development of a disease in an
individual, inhibit or slow the pment of a disease in an individual, decrease the frequency
or severity of symptoms in an individual, and/or se the recurrence in an individual who
currently has or who previously has had a disease.
The terms "prophylactic treatment” or "preventive ent" relate to any ent that is
intended to prevent a disease from occurring in an individual. The terms ”prophylactic treatment"
or "preventive treatment" are used herein interchangeably.
The terms "individual" and "subject" are used herein hangeably. They refer to human
beings, non—human primates or other mammals (eg. mouse, rat, rabbit, dog, cat, cattle, swine,
sheep, horse or primate) that can be ed with or are susceptible to a disease or er (e.g.,
cancer) but may or may not have the disease or disorder. In many embodiments, the individual is
a human being. Unless otherwise stated, the terms "individua " and “subject" do not denote a
particular age, and thus ass adults, ies, en, and newborns. In preferred
embodiments of the present invention, the "individual" or "subject" is a "patient“. The term
"patient" means according to the invention a subject for treatment, in particular a diseased
By "being at risk" is meant a subject, i.e. a patient, that is identified as having a higher than
normal chance of developing a disease, in particular cancer, compared to the general population.
In addition, a subject who has had, or who currently has, a disease, in particular cancer is a
t who has an increased risk for developing a disease, as such a subject may continue to
develop a disease. Subjects who currently have, or who have had, a cancer also have an
increased risk for cancer metastases.
The term "immunotherapy" relates to a treatment involving a specific immune reaction.
In the context of the present invention, terms such as "protect", "prevent", "prophylactic",
"preventive", or ”protective" relate to the prevention or treatment or both of the occurrence
and/or the ation of a disease in a subject and, in particular, to minimizing the chance that a
subject will p a disease or to ng the development of a disease. For example, a person
at risk for a tumor, as bed above, would be a candidate for therapy to prevent a tumor.
A prophylactic administration of an immunotherapy, for e, a prophylactic administration
of an agent or composition of the invention, preferably protects the recipient from the
development of a e. A therapeutic administration of an immunotherapy, for example, a
therapeutic administration of an agent or composition of the invention, may lead to the inhibition
of the progress/growth of the disease. This comprises the deceleration of the progress/growth of
the disease, in particular a disruption of the ssion of the disease, which preferably leads to
elimination of the disease.
Immunotherapy may be performed using any of a variety of techniques, in which agents
provided herein preferably on to remove CLDN6-expressing cells from a patient. Such
removal may take place as a result of ing or inducing an immune
response in a patient
specific for CLDN6 or a cell sing CLDN6 and/or presenting CLDN6 in the context of
MHC molecules.
Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment
relies on the in vivo stimulation of the endogenous host immune system to react t diseased
cells with the administration of immune response-modifying agents (such as peptides and nucleic
acids as provided herein).
Within other ments, immunotherapy may be passive immunotherapy, in which treatment
involves the delivery of agents with established tumor-immune reactivity (such as effector cells)
that can directly or indirectly mediate antitumor effects and does not necessarily depend
on an
intact host immune system. Examples of effector cells include T lymphocytes (such as CD8+
cytotoxic T lymphocytes and CD4+ T-helper lymphocytes), and antigen-presenting cells (such as
dendritic cells and macrophages). T cell ors specific for the CLDN6 peptides recited herein
and artificial T cell receptors specific for CLDN6 may be transferred into effector cells for
adoptive immunotherapy.
As noted above, immunoreactive peptides as provided herein
may be used to rapidly expand
antigen—specific T cell cultures in order to generate a sufficient number of cells for
immunotherapy. In particular, antigen—presenting cells, such as dendritic cells, macrophages,
monocytes, asts and/or B cells, may be pulsed with immunoreactive peptides or
transfected with one or more nucleic acids using standard techniques well known in the art.
Cultured effector cells for use in y must be able to grow and distribute widely, and to
survive long term in viva. Studies have shown that cultured or cells can be induced to
grow
in vivo and to survive long term in substantial numbers by repeated stimulation with n
supplemented with IL-2 (see, for example, Cheever et a1. (1997), Immunological Reviews 1 5 7,
177.
Alternatively, a nucleic acid expressing a peptide d herein may be introduced into antigen-
presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the
same patient.
Transfected cells may be reintroduced into the patient using any means known in the art,
preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor
administration.
Methods disclosed herein may involve the administration of autologous T cells that have been
activated in response to a peptide or peptide-expressing antigen presenting cell. Such T cells may
be CD4+ and/or CD8+, and may be erated as described above. The T cells may be
administered to the subject in an amount ive to inhibit the development of a disease.
The term "immunization“ or "vaccination" describes the process of treating a t with the
purpose of inducing an immune response for therapeutic or prophylactic reasons.
The term "in vivo" relates to the Situation in a subject.
According to the invention, a "sample" may be any sample useful according to the present
invention, in particular a biological sample such a tissue sample, including body fluids, and/or a
ar sample and may be obtained in the conventional manner such as by tissue biopsy,
ing punch biopsy, and by taking blood, bronchial aspirate, sputum, urine, feces or other
body fluids. According to the invention, the term "sample" also includes processed samples such
as fractions or isolates of biological samples, e.g. nucleic acid and peptide/protein isolates.
The compounds and agents described herein may be administered in the form of any le
pharmaceutical composition.
The pharmaceutical compositions of the ion are preferably sterile and contain an effective
amount of the agents described herein and optionally of further agents as discussed herein to
generate the desired reaction or the desired effect.
Pharmaceutical compositions are usually provided in a uniform dosage form and may be
prepared in a manner known per se. A pharmaceutical composition may e.g. be in the form of a
solution or suspension.
A pharmaceutical composition may se salts, buffer substances, preservatives, carriers,
diluents and/or excipients all of which are preferably pharmaceutically acceptable. The term
aceutically acceptable" refers to the non-toxicity of a material which does not interact
with the action of the active component of the pharmaceutical composition.
Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically
able salts and are ed in the invention. Pharmaceutically able salts of this kind
comprise in a non limiting way those ed from the following acids: hydrochloric,
hydrobromic, ic, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,
succinic acids, and the like. Pharmaceutically acceptable salts may also be prepared as alkali
metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.
Suitable buffer substances for use in a pharmaceutical composition include acetic acid in a salt,
citric acid in a salt, boric acid in a salt and oric acid in a salt.
Suitable preservatives for use in a pharmaceutical composition include benzalkonium chloride,
chlorobutanol, paraben and thimerosal.
An injectible formulation may comprise a pharmaceutically acceptable excipient such as Ringer
The term "carrier" refers to an organic or inorganic component, of a natural or synthetic nature,
in which the active component is combined in order to facilitate, enhance or enable application.
According to the invention, the term "carrier" also includes one or more compatible solid or
liquid fillers, diluents or encapsulating substances, which are suitable for administration to a
patient.
Possible carrier substances for parenteral administration are eg. sterile water, Ringer, Ringer
e, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in
particular, biocompatible lactide rs, lactide/glycolide copolymers or
polyoxyethylene/polyoxy— propylene copolymers.
The term ient" when used herein is intended to indicate all substances which may be
present in a pharmaceutical composition and which are not active ingredients such as, e.g.,
carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers,
flavoring agents, or colorants.
The agents and itions described herein may be administered via
any conventional route,
such as by parenteral administration including by injection or infusion. Administration is
preferably erally, e.g. intravenously, intraarterially, subcutaneously, ermally or
intramuscularly.
Compositions suitable for parenteral administration usually se a sterile aqueous or
nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the
recipient. Examples of compatible carriers and solvents are Ringer solution and ic sodium
chloride solution. In on, usually sterile, fixed oils are used as solution or suspension
medium.
The agents and compositions described herein are administered in effective amounts. An
"effective amount" refers to the amount which achieves a desired on or a desired effect
alone or together with further doses. In the case of ent of a ular disease
or of a
particular condition, the desired reaction preferably relates to inhibition of the course of the
disease. This ses slowing down the
progress of the disease and, in particular, interrupting
or reversing the progress of the disease. The desired reaction in a treatment of a disease
or of a
condition may also be delay of the onset or a prevention of the onset of said disease
or said
condition.
2015/056899
An effective amount of an agent or composition bed herein will depend on the ion to
be treated, the severeness of the disease, the individual parameters of the patient, including age,
physiological condition, size and weight, the duration of treatment, the type of an accompanying
therapy (if present), the specific route of administration and similar factors. Accordingly, the
doses administered of the agents described herein may depend on various of such parameters. In
the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively
higher doses achieved by a different, more localized route of administration) may be used.
The agents and compositions described herein can be administered to patients, e.g., in vivo, to
treat or prevent a variety of disorders such as those described herein. Preferred patients include
human patients having disorders that can be corrected or ameliorated. by stering the agents
and compositions described herein. This includes disorders involving cells characterized by
expression of CLDN6.
For example, in one embodiment, agents described herein can be used to treat a t with a
cancer disease, e.g., a cancer e such as described herein terized by the presence of
cancer cells expressing CLDN6.
The pharmaceutical compositions and methods of treatment described according to the invention
may also be used for immunization or vaccination to prevent a disease described herein.
The pharmaceutical composition of the invention may be administered together with
supplementing ty-enhancing substances such as one or more adjuvants and may comprise
one or more immunity-enhancing nces to further increase its effectiveness, ably to
achieve a synergistic effect of immunostimulation. The term "adjuvant" relates to compounds
which gs or enhances or rates an immune response. Various mechanisms are
possible in this respect, ing on the various types of nts. For example, compounds
which allow the maturation of the DC, e.g. lipopolysaccharides or CD40 ligand, form a first class
of suitable adjuvants. Generally, any agent which influences the immune system of the type of a
"danger signal" (LPS, GP96, dsRNA etc.) or cytokines, such as GM-CSF, can be used as an
adjuvant which enables an immune response to be intensified and/or influenced in a controlled
manner. CpG oligodeoxynucleotides can optionally also be used in this t, although their
side effects which occur under certain circumstances, as explained above, are to be considered.
Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins or
chemokines, e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, lL—8, IL—9, IL—10, IL-12, IFNa, IFNy,
GM—CSF, LT-a, or growth factors, e.g. hGH. Further known adjuvants are aluminium hydroxide,
Freund's adjuvant or oil such as Montanide®, most preferred Montanide® lSASl. Lipopeptides,
such as Pam3Cys, are also suitable for use as adjuvants in the pharmaceutical composition of the
t invention.
The pharmaceutical composition can be administered locally or systemically, preferably
systemically.
The term "systemic administration" refers to the administration of an agent such that the agent
becomes Widely distributed in the body of an individual in significant amounts and develops a
desired effect. For example, the agent may develop its desired effect in the blood and/or reaches
its desired site of action via the vascular system. Typical systemic routes of administration
e administration by introducing the agent directly into the vascular system or oral,
pulmonary, or intramuscular administration wherein the agent is adsorbed, enters the vascular
system, and is carried to one or more desired site(s) of action via the blood.
According to the t invention, it is preferred that the systemic stration is by
parenteral administration. The term "parenteral administration" refers to administration of an
agent such that the agent does not pass the intestine. The term "parenteral administration"
includes intravenous administration, subcutaneous stration, intradermal administration or
intraarterial administration but is not limited thereto.
Administration may also be carried out, for example, orally, intraperitonealy or uscularly.
The agents and compositions provided herein may be used alone or in combination with
tional eutic ns such as y, irradiation, chemotherapy and/or bone
marrow transplantation (autologous, syngeneic, allogeneic or unrelated).
The present invention is described in detail by the figures and examples below, which are used
only for illustration purposes and are not meant to be limiting. Owing to the description and the
examples, further embodiments which are se included in the invention are accessible to the
skilled worker.
2015/056899
Figure 1: Representation of the TCR-CD3 complex. The intracytoplasmic CD3
immunoreceptor tyrosine-based activation motifs (ITAMs) are indicated as cylinders (adapted
from "The T cell receptor facts book", MP Lefranc, G Lefranc, 2001 ).
Figure 2: The design of successive generations of CARS. tic representation of the
different generations of CARS (1G, first generation, 2G, second generation, 3G, third
tion). The first generation contains extracellular scFvs and the cytoplasmic CD3Z
chain/ZAP70 mediating cytotoxicity, the second. generation additionally CD28/PI3K promoting
proliferation and the third generation furthermore 4-lBB or OX40/TRAF ning cell survival
(Casucci, M. et al. (2011) 2: 378-382).
Figure 3: Schematic representation of the different receptor formats for the redirection of
T cells against CLDN6. Left: a second generation CAR consisting of a CLDN6-specific scFv
fragment, a IgGl-derived spacer domain, a CD28 costimulatory and a CD3( signaling domain
(CAR-28C); : a novel CAR format based on the linkage of the scFV with the constant
domain of the murine TCRB chain and coexpression of the constant domain of the murine TCRa
chain a); right: a murine TCR composed of TCR (1/B chains (mu, murine TCR);
Figure 4: Claudin-6 expression in normal tissues and different cancers. The CLDN6 mRNA
expression was analyzed by qRT-PCR in different normal tissue and 47 ovarian oma
specimens.
Figure 5. Technology platform for TCR isolation and validation. The approach integrates all
steps from isolation of antigen-specific T cells (top) to TCR cloning (middle) and TCR
validation (bottom). HLA—AZ/DRl-transgenic mice are immunized with tumor antigen encoding
mRNA. Spleen cells of these mice are analyzed for ex vivo reactivity against the respective
n by lFNv-ELISPOT and antigen-specific murine CD8+ T cells are isolated after in vitro
restimulation based on tion-induced sion of CD137 by flow cytometry (top). Single
cells are ted in multiwell-plates and subjected to first-strand cDNA synthesis and
enrichment by a global PCR amplification step. TCR (X/B variable regions are cloned into vectors
for in vitro transcription (IVT) containing the constant region cassettes (middle). TCR d/B chain
2015/056899
RNAs are transferred into human CD8+ T cells, cocultured with APCs expressing the
appropriate antigen and HLA les and tested for functional reprogramming of engineered
T cells (bottom).
Figure 6: Ex vivo reactivity of spleen cells from immunized HLA-A*02-transgenic mice
against CLDN6-derived peptides analyzed by IFNy—ELISPOT assay. HLA-A*02 CLDN6-
specific g peptides were predicted applying a specific algorithm (Rammensee H. et a1.
(1999) Immunogenetics 50, 213-9). Spleen cells were analyzed for reactivity against CLDN6
peptide pool or predicted HLA-A*02-binding CLDN6-derived es . Positive control:
PMA-treated spleen cells; ve l: an irrelevant peptide pool (HIV-gag), irrelevant
nonamer peptide (PLAC13 9).
Figure 7: Flow cytometry sorting of CLDN6-specific murine CD8+ T cells from HLA—
A*02-transgenic mice after in-vitro restimulation. Single CD8+/CD137+ T cells were ed
by flow cytometry and harvested in multiwell plates for TCR g after restimulation of
spleen cells with CLDN6 overlapping e pool. Control: Spleen cells restimulated with
irrelevant peptide pool.
Figure 8: Specificity testing of TCRs isolated from CD8+ T cells of CLDN6-immunized
mice. CD8+ T cells of a HLA-A*O2—positive healthy donor were transfcted with TCR-a/B chain
RNAs and tested for recognition of K562~A2 cells transfected with CLDN6 RNA or pulsed with
CLDN6 overlapping 15mer peptides (= C16 pool) or CLDN6 HLA—A*02 binding peptides (C16-
A2-1, Cl6—A2-2) by IFNy—ELISPOT. Negative controls: irrelevant peptide pool, irrelevant 9mer
peptide; ve control: SEB
Figure 9: Surface expression of CLDN6-specific murine TCRs on human preactivated
CD8+ T cells. CD8+ T cells were preactivated with OKT3 and transfected with 20
pg TCR a/B
RNA. 20h after electroporation cells were stained with a PE-conjugated anti—CD8 antibody and
AFC-conjugated antibody recognizing the murine constant domain of the TCR B chain. Cells
were gated on single cytes.
Figure 10: Tumor cell lysis mediated by CLDN6—specific TCRs. Preactivated CD8+ T cells
were transfected with ZOpg TCR (it/B RNAs and cocultured 20h later together with HLA-A*02-
expressing CLDN6-positive (PAl—Luc; NIH-OvCar3) or ~negative (SK-Mel—37) tumor cell lines
with an E:T tor cell : target cell) ratio of 30:1. Specific lysis was analyzed by luciferase—
based cytotoxicity assay after 4h ure.
Figure 11: Dose-dependent eration mediated by CLDN6—specific TCRs in response to
CLDNG-expressing target cells. CD8+ T cells were transfected with 20ug TCR RNA, labeled
with CFSE and cocultured with autologous monocytes transfected with titrated amounts of
CLDN6 RNA. After 4 days of coculture cells were stained With an APC-Cy7-labeled anti-CD8
antibody. A) c proliferation was analyzed by flowcytometry based on the dilution of the
CFSE proliferation dye. Dotplots show living CD8+ T lymphocytes after coculture with
monocytes transfected with lug CLDN6-RNA. B) Bars show the percentage of proliferating
CD8+ T cells.
Figure 12: Surface expression of CLDNG—specific CAR constructs on resting human CD4+
and CD8+ T cells. PBMCs were transfected with lOug CAR RNA. 20h after electroporation
cells were stained with a njugated anti-CD8, a FITC-conjugated anti-CD4 and an idiotype-
c antibody labeled with Dylight-650. Cells were gated on single CD4+ or CD8+ T cells.
Figure 13: Tumor cell lysis mediated by different CLDN-6 targeting receptor formats.
Preactivated CD8+ T cells were transfected with CAR or TCR RNAS and cocultured 20h later
together with positive or CLDN6—negative tumor cell lines PAl and
Luc at different E:T ratios. Specific lysis was ed by luciferase-based xicity assay
after 4h coculture.
Figure 14: Antigen-specific proliferation mediated by CLDNG-specific CAR in response to
CLDNG-expressing target cells. CD8+ T cells were transfected with 20ug TCR or CAR RNA,
d with CFSE and cocultured with autologous iDC transfected with CLDN6 or control RNA
for 4 days. A) TCR/ CAR surface expression was analyzed by flow cytometry after staining with
a murine AFC—conjugated TCRB-specific or a DylightGSO-conjugated idiotype-specific antibody.
Specific proliferation was analyzed by flow cytometry based on the dilution of the CFSE
proliferation dye.
Figure 15: Surface expression of different mutants of CLDN6-CAR—28Z constructs with
mutated cysteine 46 on preactivated CD8+ T cells. CD8+ T cells were preactivated with
OKT3 and transfected with 20ug CAR RNA. 20h after electroporation cells were stained with a
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PIE—conjugated anti—CD8 antibody and an idiotype-specific antibody labeled with t650.
Cells were gated on singlets and lymphocytes.
Figure 16: Surface expression of different mutants of CAR—28: constructs with d
cysteine 46 on preactivated CD8+ T cells of three different donors. CD8+ T cells were
preactivated with OKT3 and transfected with ZOug CAR RNA. 20h after electroporation cells
were stained with an idiotype-specific antibody labeled with Dylight650. Cells were gated on
CAR—expressing CD8+ T lymphocytes. The results of three independent experiments are shown.
Top: the percentage of CAR+/CD8+ T cells is shown; bottom: the mean fluorescence intensity of
CAR-positive CD8+ T cells is shown;
Figure 17: Specific tumor cell lysis mediated by different s of CLDN6-CAR-28C
constructs with mutated cysteine 46. A) The CLDN6 e expression on target cell lines
was analyzed after staining with a Alexa647-conjugated CLDN6-specific antibody by flow
cytometry. B) Preactivated CD8+ T cells were transfected with 20ug CAR RNA and cocultured
20h later together with CLDN6—positive (PAl) or CLDN6—~negative (MDA—MBLuc~
Tomato) tumor cell lines at different E:T ratios. Specific lysis was analyzed by rase-based
cytotoxicity assay after 4h ure. C) CAR surface sion on T cells was analyzed after
staining with a fluorochrome-conjugated CD8-specific and an idiotype-specific antibody by flow
cytometry.
Figure 18: Dose-dependent lysis of target cells mediated by different mutants of CLDN6-
CAR—28C constructs with d cysteine 46. A) Preactivated CD8+ T cells were transfected
with 20ug CAR RNA and cocultured 20h later together with autologous iDC transfected with
titrated amounts of CLDN6~RNA (E:T = 30:1). B) The CLDN6 surface expression on
transfected iDCs was analyzed after staining with a Alexa647—conjugated CLDN6-specific
antibody by flowcytometry.
Figure 19: Schematic representation of the retroviral SIN construct used for stable CAR
expression. The plasmid pESlZ.6-CLDN6-CAR-C46S was used for transient tion of
GALV—enveloped SIN-vector using T cells.
Figure 20: Detection of CLDN6-CAR and CAR against an unrelated tumor antigen on
transduced human T cells used for adoptive transfer into NSG mice. Cells were stained with
fluorochrome—conjugated antibodies (BD Biosciences) directed against CD8 and CD4 as well as
with idiotype-specific antibodies directed t the respective scFv part of the CLDN6-CAR
(anti-IMAB206, d Pharmaceuticals AG) and the CAR against an unrelated tumor
n, respectively. Cells were gated on single CD8+ or CD4Jr lymphocytes. Transduced T cells
were used for adoptive cell transfer in C12-engrafted NSG mice. The transduction rate
for the CLDN6-CAR and the CAR against an unrelated tumor antigen was about 37% of CD4+
and 20% of CD8+ as well as 36% of CD4+ and 24 of CD8+ cells, respectively. Graphs are
displayed in logarithmic scale.
Figure 21: Anti—tumoral activity of CLDN6—CAR uced T cells in an ovarian
carcinoma model. 1x107 human OV90~SC12 tumor cells (ATCC CRL11732) were injected
subcutaneously into NSG mice (10 mice/ group). After 4 days, the mice were treated with a
single intravenous injection of lxlO7 28 bead stimulated, irally uced human
T cells (about 37% of CD4 and 20% of CD8 were CLDN6-CAR positive). A) Scheme of the
experimental set up. B) Delay of tumor growth in CLDN6-CAR treated mice compared to
control groups (no T cells, untransduced T cells, and T cells transduced with CAR against an
unrelated tumor n). Tumor monitoring by volume measurements and analysis of peripheral
blood was performed weekly. Results are expressed as mean tumor volume i SEM with n=10
mice for all groups. Tumor volume was calculated using the following formula:
V=1/2*(length*square width). The plot for the CLDN6-CAR treated mice is significantly
different from the control treatment group for t=3 ldays (*ANOVA, P<0.05). C) Tumor—growth
curves of the individual mice of each group are shown. Please note, 2 mice in the unrelated
tumor antigen group had to be ced on day 24 due to high tumor burden (marked with +).
Figure 22. Proliferation of CAR T cells after co-culture with CLND6 expressing iDCs.
CD8+ T cells were transfected with IVT-RNA encoding a CAR directed against A) CLDN6 or
B) an unrelated tumor antigen as negative control, labeled with CFSE (carboxyfluorescein
succinimidyl ester) and cocultured with CLDN6-transfected autologous iDCs for 4 days.
Proliferation of CAR T cells was analyzed based on the dilution of CFSE by ometry.
Cells were gated on single living CD8+ T lymphocytes.
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EXAMPLES
The ques and methods used herein are described herein or d out in a manner known
per se and as bed, for example, in Sambrook et a1., lar Cloning: A tory
Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All
methods including the use of kits and reagents are carried out according to the manufacturers’
information unless specifically indicated.
e 1: Materials and Methods
Cell lines and reagents
The human chronic myeloid leukemia cell line K562 (Lozzio, C.B. & Lozzio, BB (1975), Blood
45, 321-334) was cultured under standard conditions. K562 cells stably transfected with HLA-
A*0201 (Britten, C.M. et a1. (2002), J. Immunol. Methods 259, 95-110) (referred to eg. as
K562-A*0201) were used for validation assays. The primary human newborn foreskin fibroblast
cell line CCD-1079Sk (ATCC No. CRL-2097) was cultured according to the manufacturers’
instructions.
The human CLDN6 expressing ovarian carcinoma cell line OV—90-SC12 was used for in Vivo
validation of the CLDN6-CAR.
The culture medium for PA-l-SC12_A0201_luc_gfp_F7 is composed of 86% RPMI 1640+
Glutamax (Co. Gibco, Cat—No. 61870), 10% FCS (Co. Biochrome, Cat-No. $0615), 1% Sodium
Pyruvate (lOOmM) (Co. Gibco, Cat—No. 11360), 1% MEM Non~Essential Amino Acids Solution
(IOOX) (Co. Gibco, Cat—No. 11140), 2% Sodium Bicarbonate 7,5% solution (Co. Gibco, Cat-No.
25080).
The culture medium for OVSC12 is composed of 41,5% MCDB 105 (C0. Sigma Aldrich,
Cat-No. M6395—1L), 41,5% Medium 199 (C0. Sigma Aldrich, Cat—No. M2154-500mL), 15%
FCS (Co. Biochrome, Cat-No. 80615), 2% Sodium onate 7,5% solution (Co. Gibco, Cat-
No. 25080).
The culture medium for SK-MEL-37 is composed of 90% DMEM+ Glutamax (Co. Gibco, Cat-
No. 31966), 10% FCS (Co. Biochrome, Cat-No. SO615).The culture medium for MDA—MB—
c_tom is ed of 88% RPMI 1640+ Glutamax (Co. Gibco, Cat-No. 61870), 10%
FCS (Co. Biochrome, Cat-No. 80615), 1% Sodium Pyruvate (lOOrnM) (Co. Gibco, .
11360), 1% MEM Non-Essential Amino Acids Solution (100K) (Co. Gibco, Cat—No. 11140).
Feeding and/ or splitting of the cell lines was done every 2 to 3 days.
eral blood mononuclear cells ), monocytes and dendritic cells (DCs)
PBMCs were isolated by Ficoll—Hypaque (Amersham Biosciences, Uppsala, Sweden) density
gradient centrifugation from buffy coats. HLA allelotypes were determined by PCR standard
methods. tes were enriched with anti-CD14 microbeads nyi Biotech, Bergisch-
Gladbach, Germany). Immature DCs (iDCs) were obtained by differentiating monocytes for 5
days in cytokine-supplemented culture medium as described in Kreiter et a1. (2007), Cancer
Immunol. ther., CH, 56, 1577—87.
Peptides and peptide g of stimulator cells
Pools ofN- and C—tenninally free lS-mer peptides with 11 amino acid overlaps corresponding to
sequences of Claudin-6 or HIV-gag (referred to as antigen peptide pool) were sized by
standard solid phase chemistry (JPT GmbH, Berlin, Germany) and ved in DMSO to a final
concentration of 0.5 mg/ml. Nonamer peptides were reconstituted in PBS 10% DMSO. For
pulsing stimulator cells were ted for l h at 37 °C in culture medium using ent
peptide concentrations.
Vectors for in vitro transcription (IVT) of RNA
All constructs are ts of the previously described pSTl-sec-insert-ZBgUTR-A(120)—Sapl
plasmid (Holtkamp, S. et al. (2006), Blood 108, 4009-4017). To obtain ds encoding
human TCR , cDNA coding for TCR-0t or TCR-131 and TCR—Bz constant regions were
amplified from human CD8+ T cells and cloned into this backbone. For generation of plasmids
encoding murine TCR chains, cDNAs coding for TCR-(1, —[31 and —[32 constant regions were
ordered from a commercial provider and cloned analogously (GenBank accession numbers
M14506, M64239 and X67127, respectively). Specific V(D)J PCR products were introduced
into such cassettes to yield full—length TCR chains (referred to as pSTl-human/murineTCRdB-
215gUTR—A(120)).
Analogously, individual HLA class I and II alleles cloned from PBMCs of donors and beta
microgobulin (82M) cDNA from human DCs were inserted into this backbone (referred to as
pSTl-HLA class I/II-ZBgUTR-A(120) and pSTl-BZM—ZBgUTR-A(1 20)).
Plasmids coding for pp65 antigen of CMV (pSTl-sec-pp65-MITD—2l3gUTR-A(120)) and NY—
ESO-I (pSTl—sec-NY-ESO-l-MITD-ZBgUTR-A(120)) linked to a secretion signal (sec) and the
MHC class I trafficking signal (MITD) were described previously (Kreiter, S. et al. (2008), J.
Immunol. 180, 309-318). PLACl encoding d pSTl~sec-PLAC1-MITD-2l3gUTR—A(120)
was generated by cloning a cDNA obtained from a commercial provider (GenBank accession
number NM_021796) into the Kreiter et a]. backbone. TPTE encoding plasmids pSTl-agUTR—
TPTE—ZfigUTR-AUZO) and pSTl-0LgUTR—TPTE—MITD-ZBgUTR-A(l20) were generated by
cloning a cDNA obtained from a commercial provider (GenBank accession number AF007118)
into a variant of the Holtkamp el‘ a]. vector featuring an additional globin 5’-untranslated
region.
Primers were purchased from Operon Biotechnologies, Cologne, Germany.
Generation of in vitro transcribed (IVT) RNA and transfer into cells
Generation of IVT RNA was performed as described previously (Holtkamp, S. et a1. (2006),
Blood 108, 4009-4017) and added to cells suspended in X-VIVO 15 medium , Basel,
Switzerland) in a pre-cooled 4-mm gap sterile electroporation cuvette (Bio-Rad Laboratories
GmbH, Munich, Germany). Electroporation was med with a Gene-Pulser-Il apparatus
(Bio-Rad Laboratories GmbH, Munich, y) (T cells: 450 V/250 uF; IVSB T cells: 350
W200 uF; SupTl (ATCC No. CRL—1942): 300 W200 uF; human DC: 300 W150 uF; K562: 200
W300 uF).
In vivo priming of T cells by intranodal immunization of HLA A2.1/DR1 mice with IVT
T cells of AZ/DRl mice (Pajot A. et al. (2004), Eur. J. lmmunoi. 34, 3060-69) were primed in
vivo against the antigen of interest by repetitive intranodal immunization using n-encoding
IVT RNA (Kreiter S. et al. (2010), Cancer ch 70, 9031-40). For odal immunizations,
mice were anesthetized with xylazine/ketamine. The inguinal lymph node was ally
exposed, 10 uL RNA (20ug) diluted in Ringer’s solution and Rnase—free water were injected
slowly using a single-use 0.3-ml syringe with an ultrafine needle (31G, BD ences), and the
wound was closed. After six immunization cycles the mice were sacrificed and spleen cells were
isolated.
Harvest of spleen cells
Following their dissection under sterile conditions, the spleens were transferred to PBS
containing falcon tubes. The spleens were mechanically disrupted with forceps and the cell
suspensions were obtained with a cell strainer (40 um). The splenocytes were washed with PBS
centrifuged and ended in a hypotonic buffer for lysis of the erythrocytes. After 5 min
incubation at RT, the reaction was stopped by adding 20-30 ml medium or PBS. The spleen cells
were centrifuged and washed twice with PBS.
Single-cell sorting of antigen-specific CD8+ T cells after CD137 staining
For antigen-specific restimulation 2.5xlOA6/well spleen cells from zed A2/DR1 mice
were seeded in a l plate and pulsed with a pool of overlapping es encoding the
antigen of interest or a control antigen. After 24 h incubation cells were harvested, stained with a
FITC—ccnjugated anti-CD3 antibody, a PIE-conjugated anti-CD4 dy, a PerCP-Cy5.5-
conjugated anti-CD8 antibody and a Dylight—649-conjugated anti—CD137 antibody. Sorting was
ted on a BD FACS Aria flow cytometer (BD Biosciences). Cells positive for CD137,
CD3 and CD8 were sorted, one cell per well was harvested in a 96—well V—bottom-plate (Greiner
e) containing human CCD—1079Sk cells as feeder cells, centrifuged at 4 OC and stored
immediately at —80 OC.
RNA extraction, SMART—based cDNA synthesis and unspecific amplification from sorted
cells
RNA from sorted T cells was extracted with the RNeasy Micro Kit (Qiagen, Hilden, Germany)
according to the instructions of the supplier. A d BD SMART protocol was used for
cDNA synthesis: BD PowerScript Reverse Transcriptase (BD Clontech, Mountain View, CA)
was combined with Oligo(dT)-T-primer long for priming of the first-strand synthesis reaction and
TS-short (Eurogentec S.A., Seraing, Belgium) introducing an Oligo(riboG) sequence to allow for
creation of an ed template by the terminal transferase ty of the reverse transcriptase
and for template switch (Matz, M. et a1. (1999) Nucleic Acids Res. 27, 1558-1560). First strand
cDNA sized according to the manufacturer’s instructions was subjected to 21 cycles of
amplification with 5 U Pqultra Hotstart High—Fidelity DNA Polymerase (Stratagene, La Jolla,
CA) and 0.48 uM primer TS—PCR primer in the presence of 200 uM dNTP (cycling ions: 2
min at 95 °C for, 30 s at 94 °C, 30 s at 65 °C, 1 min at 72 °C for, final extension of 6 min at 72
°C). Successful amplification of TCR genes was controlled with either human or murine TCR-B
constant region specific primers and consecutive clonotype—specific human or murine VOL-NB—
PCRs were only performed if strong bands were detected.
First strand cDNA for the amplification of HLA class I or 11 sequences was synthesized with
SuperScriptII Reverse Transcriptase (Invitrogen) and Oligo(dT) primer with 1-5 ug RNA
ted from patient-derived PBMCs.
Design of PCR primers for TCR and HLA amplification
For design of human TCR consensus primers, all 67 TCR-VB and 54 TCR-Vet genes (open
reading frames and pseudogenes) as listed in the ImMunoGeneTics (IMGT) database
(http://www.imgtorg) together with their corresponding leader sequences were aligned with the
BioEdit ce Alignment Editor (e.g. http://www.bio-soft.net). Forward primers of 24 to 27
bp length with a maximum of 3 degenerated bases, a GC-content between 40-60% and a G or C
at the 3’end were designed to anneal to as many leader sequences as le and ed with
a 15 bp S’extension featuring a rare ction enzyme site and Kozak sequence. Reverse
primers were designed to anneal to the first exons of the constant region genes, with primer
1_as g to sequences corresponding to amino acids 7 to 16 of Cd and TRBCex1_as
to amino acids (aa) 8 to 16 in C81 and C132. Both oligonucleotides were synthesized with a 5’
phosphate. Primers were bundled in pools of 2—5 forward oligos with identical annealing
temperature.
This strategy was replicated for the design of murine TCR consensus primers, aligning 129 listed
TCR-VOL and 35 listed TCR-VB genes. Reverse primers mTRACexJWas and mTRBCex1_aS are
homologous to sequences corresponding to aa 24 to 31 and 8 to 15, respectively.
HLA consensus primers were designed by aligning all HLA class I and II sequences listed on the
Anthony Nolan Research Institute website (www.anthonynolan.com) with the BioEdit ce
Alignment . Forward primers of 23 to 27 bp length with a maximum of 3 degenerated but
reserving bases annealing to as many as possible HLA sequences of one locus were
equipped with a sphate and Kozak sequence extension. Reverse primers were designed
analogously but without uction of wobble bases and equipped with a 14 bp 5’-extension
encoding an AsiSI restriction enzyme site.
PCR amplification and g of V(D)J sequences
3-6 pl of preamplified cDNA from ed T cells was subjected to 40 cycles of PCR in the
ce of 0.6 uM Va-lVB-specific oligo pool, 0.6 pM Ca- or CB—oligo, 200 ”M dNTP and 5 U
Pfu polymerase (cycling conditions: 2 min at 95 °C, 30 s at 94 °C, 30 s annealing temperature, 1
min at 72 °C, final extension time of 6 min at 72 °C). PCR products were analyzed using
Qiagen’s capillary electrophoresis system. Samples with bands at 400-500 bp were size
fractioned on agarose gels, the bands excised and purified using a Gel Extraction Kit (Qiagen,
Hilden, Germany). Sequence analysis was performed to reveal the sequence of both the V(D)J
domain and [3 constant region, as TRBCex1_as and mTRBCex1_as primer, respectively, match to
both TCR constant region genes [31 and 62 in human and mouse, respectively. DNA was
digested and cloned into the WT vectors containing the appropriate backbone for a complete
TCR-d/B chain.
Flow cytometric analyses
Cell e expression of ected TCR genes was analyzed by flow try using PE-
conjugated anti-TCR antibody against the appropriate variable region family or the constant
region of the TCR [3 chain (Beckman r Inc., Fullerton, USA) and FITC-/APC-labeled anti-
CD8/-CD4 antibodies (BD Biosciences). Cell e expression of transfected CARS was
analyzed using a Dylight-6SO-conjugated idiotype-specific antibody (Ganymed ceuticals)
recognizing the scFv fragment contained in all CLDN6—CAR constructs. HLA antigens were
detected by staining with FITC—labeled HLA class Il-speciflc (Beckman Coulter Inc., ton,
USA) and PE-labeled HLA class I-specific dies (BD Biosciences). CLDN6 surface
expression on target cells was analyzed by staining with an Alexa-Fluor647-conj ugated CLDN6-
specific dy (Ganymed Pharmaceuticals). Flow cytometric analysis was performed on a
FACS CANTO II flow cytometer using the FACS Diva software (BD Biosciences).
Luciferase cytotoxicity assay
For assessment of cell-mediated cytotoxicity a bioluminescence-based assay was established as
an alternative and optimization to 51Cr release. In contrast to the standard chromium release
assay, this assay measures lytic ty of eifector cells by calculating the number 01 viable
luciferase expressing target cells following coincubation. The target cells were stably or
transiently transfected with the luciferase gene coding for the firefly luciferase from firefly
Photinus pyralis (EC 1.13.127). Luciferase is an enzyme catalyzing the oxidation of luciferin.
The reaction is ATP-dependent and takes place in two steps:
luciferin + ATP ——> luciferyl ate + PE
luciferyl adenylate + Q; ——> oxyluciferin + AMP + light
Target cells were plated at a concentration of 104 cells per well in white 96—well plates (Nunc,
Wiesbaden, Germany) and were cocultivated with varying numbers of TCR-transfected T cells
in a final volume of 100 pl. 3 h later 50 u] of a D-Luciferin (BD Biosciences) containing reaction
mix (Luciferin (l ug/ul), HEPES-buffer (50 mM, pH), ine 5’-triphosphatase (ATPase,
0.4 mU/ul, Sigma-Aldrich, St. Louis, USA) was added to the cells. By addition of ATPase to the
reaction mix luminescence resulting from luciferase released from dead cells was diminished.
After a total incubation time of 4 h bioluminescence emitted by viable cells was measured using
the Tecan Infinite 200 reader (Tecan, Crailsheim, Germany). Cell—killing activity was calculated
in regard to luminescence values obtained after complete cell lysis induced by the addition of 2%
Triton-X 100 and in relationship to luminescence d by target cells alone. Data output was
in counts per second (CPS) and percent specific lysis was ated as follows:
Sexp ~ CPSmm)/(CPSmax — CPSmin)» * 100.
Maximum luminescence (maximum counts per second, CPSmax) was assessed after incubating
target cells without effectors and minimal luminescences (CPSmin) was assessed after treatment
of targets with detergent Triton—X-IOO for complete lysis.
ELISPOT (Enzyme-Linked ImmunoSPOT assay)
Microtiter plates (Millipore, Bedford, MA, USA) were coated overnight at room temperature
with an anti-IFNY antibody l-le (Mabtech, Stockholm, Sweden) and blocked with 2% human
albumin (CSL Behring, Marburg, Germany). 2-5x104/well antigen presenting stimulator cells
were plated in triplicates together with 0.3—3x105/well TCR-transfected CD4+ or CD8+ effector
cells 24 h after electroporation. The plates were ted ght (37 °C, 5% C02), washed
with PBS 0.05% Tween 20, and incubated for 2 hours with the FNY biotinylated mAB 7-
86-1 (Mabtech) at a final concentration of 1 ug/ml at 37 °C. Avidin-bound horseradish
peroxidase H (Vectastain Elite Kit; Vector Laboratories, Burlingame, USA) was added to the
wells, incubated for 1 hour at room temperature and developed with 3-aminoethyl carbazole
(Sigma, hofen, y).
CFSE (Carboxyfluorescein succinimidyl ester) proliferation assay
CD8+ T cells were transfected with TCR or CAR RNA and labeled with 2.5 uM CFSE. Labeled
T cells were washed and cocultured with ansfected autologous monocytes or iDCs (E:T
(effector cells : (tumor) cells) = 10:1). After 4 days of coculture cells were harvested and
proliferation was analyzed by flow cytometry based on the progressive halving of CFSE
fluorescence within daughter cells following cell divisions.
Retroviral construct for stable CAR expression
For stable expression of the CLDN6-CAR or the CAR against an unrelated tumor n used
as a ve control the retroviral SIN vector E8126 was used e 19).
Transduction of human T cells
For the mouse adoptive cell transfer (ACT) ments, human T lymphocytes were enriched
from PBMCS of healthy donors by removal of monocytes after 2h of plastic adherence. T
lymphocytes were cultured in X—VivolS (Lonza) medium mented with 5% human AB
serum (Invitrogen), 100 U/ml 1L2 (Proleukin S, Novartis), 20 ng/ml 1L7 (Miltenyi), 10 ng/ml
IL15 (Miltenyi) and stimulated with ic anti-CD3/anti—CD28 beads (Dynabeads;
Invitrogen) at a 1:3 CD3 cell to bead ratio and transduced on days 3 and 4 post stimulation with
retroviral supernatants. Cells were expanded in X-VivolS medium supplemented with 5%
human AB serum, 300 U/ml ILZ, 20 ng/ml 1L7 and 10 ng/ml IL15. Incubation 37 °C, 5 % C029
95 0/o rH (Figure 20).
Mouse model for in-vivo validation of antitumoral activity
Xenograft tumors were established by subcutaneous ion of 1x107 OV90-SC12 human
ovarian tumor cells into 8-14 ld NODCg-Prkdcscid Iergtmlel/Szl mSG) mice (The
Jackson Laboratory, Bar Harbor, ME). After 4 days, mice were treated with a single intravenous
injection of 1x107 of CAR transduced T cells (20—37 % CAR positive). Tumor monitoring was
performed weekly by volume measurements using caliper (Figure 21(a)).
Example 2: Isolation of high-affinity HLA-A*02-restricted murine TCRs specific for
Claudin-6
We validated the immunogenic potential of CLDN6 in AZ/DRI mice by tive intranodal
immunization with CLDN6 encoding IVT-RNA and used spleen cells of these mice for isolation
of specific T cells and subsequent cloning of the corresponding TCR genes (Figure 5).
Spleen cells of immunized mice were analyzed for the successful induction of CLDN6—specific T
cells and their reactivity against predicted HLA—A*02 binding CLDN6 peptides ex-vivo by IFNy-
ELISPOT assay (Figure 6),
Significant frequencies of CLDN6-specific T cells could be induced In all three mice by RNA
immunization, whereas T cell vity was focused on two CLDN6 peptides predicted, that
were with the best HLA-A*02 g score (C16-A2-1 and Cl6-A2~2).
For isolation of CLDN6-specific T cells, spleen cells of immunized mice were restimulated in-
vitro and sorted by flow cytometry based on the activation-induced upregulation of CD137
(Figure 7).
specific CD8+ T cells could be retrieved from all three immunized A2/DR1 mice and a
total of ii CLDN6—specific TCRs were cloned from single-sorted murine T cells.
TCRs were subjected to immunological validation assays, which ed that six CLDN6-TCRs
recognized the HLA—A*0201-restricted epitope CLDN699 (C16-A2-l) and four CLDN6-
TCRs were specific for CLDN622 (Clé-AZ-Z), whereas both epitopes were previously
identified by ex-vivo ELISPOT analysis (Figure 8). One TCR (TCchg-CLDN6#7)
recognized the peptide CLDN615 (Cl6-A2-3).
Example 3: Comparative testing of murine TCRs specific for CLDN6 91-99
In total six murine TCRs were identified that all recognize the HLA-A*02-restricted epitope
CLDN699. In order to confirm that this epitope is naturally processed and presented by
endogenously CLDN6 expressing tumor cell lines and to evaluate the potential of the identified
murine TCRs to mediate killing of such cells a luciferase-based cytotoxicity assay was
performed. Human preactivated CD8+ T cells were transfected with TCR RNA and. surface
expression was analyzed by flow cytomtry (Figure 9). All murine TCRs were sed on a
high percentage of human CD8+ T cells after RNA transfer as indicated by staining with an
fluorochrome-conjugated antibody specific for the constant domain of the murine TCR—B chain.
ansfected T cells were subjected to rase-based xicity assay together with the
CLDN6—expressing tumor cell lines PAl (teratoma) and NIH-Ovcar3 (ovarian carcinoma). The
CLND6-negative breast cancer cell line MDA—MB-231 served as negative control. All TCRs
mediated efficient lysis of CLDN6-expressing tumor cell lines ranging from 38-94% of PAl and
29-76% of NIH-Ovcar3, while no lysis could be observed with sfected T cells (Figure 10).
Most target cells were lysed when the mTCchg-CLDN6 #1, #8 or #18 were used.
In order to analyze, if the murine TCRs can mediate specific proliferation of human T cells after
coculture with autologous antigen-expressing target cells a CFSE eration assay was
med (Figure ll). TCR—transfected CD8+ T cells were cocultured with autologous
monocytes transfected with titrated amounts of CLDN6 RNA. All TCRs ed Specific
proliferation indicated by the dilution of the CFSE eration dye after 4 days of coculture
with CLDN6-RNA-transfected CD14+ cells, whereas again s—CLDN6 #1, #8 or #18
showed the best results, especially when low s of CLDN6 RNA were transfected into the
target cells. We decided to use mTCRch-CLDN6 #18 as a gold standard for the lead structure
selection together with CLDN6 targeting CAR formats.
Example 4: Generation and in-vitro validation of Claudinspeeific CARS
We evaluated two different CAR formats to specifically target CLDN6 on CLDN6 expressing
target cells. One of them represents a novel format based on the linkage of the scFv with the
constant domain of the murine TCRB chain and coexpression of the constant domain of the
TCRor chain (CAR/Cor) (Voss RH et al., (2011) Molecular y 19, supplement, SS6) (Figure
3). The second format represents a classical 2nd generation CAR (CAR-28E) that contains the
signaling and costimulatory moieties of CD3: and CD28, respectively. A on of the lck
binding moiety in the CD28 endodomain abrogates 1L2 secretion upon CARengagement to
prevent induction of regulatory T cells (Kofler D.M. et al., (2011) Molecular Therapy 19 (4),
760—767). A modification of the IgGl Fc r’ domain in the extracellular moiety of the CAR
avoids ‘off-target’ activation and unintended initiation of an innate immune
response (“ombach
A. et al., (2010) Gene Therapy 17, 1206—1213).
As CARS provide HLA independent scFV—mediated antigen—binding they are functional in both
CD4+ and CD8+ T cells. Therefore, we first analyzed the CAR surface expression on CD4+ and
CD8+ T cells after transfection of CAR RNA into bulk PBMCs.
Both, the novel CAR/Cor and the classical 2nd generation CAR (CAR—28C) are expressed on the
surface of human T cells after RNA er (Figure 12). The CAR—28c was significantly better
sed on the surface of CD4+ and CD8+ T cells than the CAR/Ca. The latter one was
transferred either by cotransfection of the CAR and the Cor chain or as a 2A peptide-based
bicistronic vector for simultaneous expression of CAR and Ca genes. Flow cytometry is
demonstrated that the 2A-based linkage of CAR and Ca results in sed surface expression
compared to coexpression of the two components. As a bicistronic vector would be used for
clinical testing the linkage of the two CAR/Ca components has to be flirther ed.
To analyze the c tumor cell lysis mediated by the different CLDN6-targeting receptor
formats a luciferase-based cytotoxicity assay was performed. CAR— or TCR—transfected
preactivated CD8+ T cells were cultured with CLDN6-positive or negative tumor cell lines at
different effector-to—target ratios and the specific lysis was calculated after 4h of coculture
(Figure 13). All CAR- and TCR-transfected T cells demonstrated. significant specific lysis of
CLDN6 expressing tumor cell lines compared to untransfected T cells.
A prerequisite for the eration and persistence of CAR-engineered T cells in the patient is
the presence of antigen as demonstrated by promising clinical trial results of CD19-specific
CARs in hematologic malignancies. In y to the expansion of endogenous T cells by RNA
immunization, we wanted to analyze, if CAR T cells could also be expanded using RNA-
vaccination of target cells to provide natural CLND6 for CAR T cell stimulation. An in vitro
proliferation assay was performed using CAR-transfected CD8+ T cells together with CLDN6 or
control RNA-transfected autologous iDCs (Figure 14). The mTCchg-CLDN6 #18 mediated
best proliferation in response to CLDN6—transfected target cells (73%). The CLDN6-CAR-28Z
also resulted in a significant proportion of proliferating T cells (44%), while the CLDN6-
CAR/Cor failed to induce proliferation probably due to the lack of CD28-mediated costimulation.
As induction of proliferation is a prerequisite for successful antitumoral activity, we decided to
use CAR-28K format for further lead structure selection.
e 5: CLDN6-CAR—28Z lead structure ion for nical and clinical testing
The CLDN6-CAR=28( scFv fragment that is responsible for antigen recognition contains an
unpaired cysteine. As such a free cysteine could result in ding of the CAR protein under
certain circumstances or in ed interactions with other cyteines by the formation of
disulfide bonds, we decided to eliminate this cysteine and ged it by a serine, a glycine or
an alanine.
We than comparatively analyzed the resulting CLDN6-CAR—28( constructs regarding surface
sion (Figure 15, 16) and cytotoxicity (Figure 17). Except of the e variant all mutated
constructs demonstrated surface expression and lysis comparable to the wild-type variant.
In order to compare the affinity of the mutated CAR constructs their cytotoxic potential in
response to gous iDCs ected with titrated amounts of CLDN6 RNA was analyzed.
Even extremely little amounts of CLDN6 RNA (0.001ug) resulted in significant lysis of target
cells mediated by all CAR constructs. As the serine variant of the CLDN6-CAR-28C showed
slightly better results ing e expression and cytotoxicity, we decided to use this
variant for preclinical testing.
Example 6: In-vivo antitumoral activity of the CAR
After having determined the antitumor activity against CLDN6 expressing tumor cell lines in—
vitro the mor ability in tumor-bearing mice was determined. ore, the potency of
CLDN6-CAR uced human T cells was compared to T cells transduced with a l CAR
against an unrelated tumor antigen and untransduced T cells in a xenograft model. A total of
1x107 cells of the human ovarian carcinoma cell line OV90—SC12 were injected subcutaneously
in NSG mice. Four days after tumor engraftment the mice were treated with a single intravenous
ion of 1X107 of CAR—transduced T cells. Tumor monitoring was performed weekly by
volume measurements using caliper. Treatment of the mice with CLDN6—CAR-transduced T
cells significantly slowed tumor growth compared to l groups treated with unrelated tumor
antigen-CAR—transduced, untransduced T cells or a group not receiving T cells (Figure 21 (b)
and (c)).
Example 7: In-vitro proliferation of CLDN6-CAR T cells in response to CLDN6-expressing
target cells
In analogy to the expansion of endogenous T cells by RNA immunization, the stimulation and
expansion of CAR T cells using RNA-vaccination of target cells to provide natural CLND6 was
analyzed by in vitro proliferation assay. CD8+ T cells were transfected with IVT-RNA encoding
a CAR against CLDNS or an unrelated tumor antigen as negative control, labeled with CFSE
(carboxyfluorescein succinimidyl ester) and cocultured with CLDN6—transfected autologous
iDCs for 4 days (Figure 22). The CLDN6—CAR mediated proliferation of nearly all CD8+ T cells
in response to CLDN6-transfected iDC could be observed (95%), while only background
proliferation (1.5%) could be observed for unrelated tumor antigen-CAR transfected T cells
indicating that proliferation was not ing on the CAR ne but was CLDN6-specific.
CLDN6-specific T cell epitopes
A2-1 (aa 91-99)
ALFGLLVYL
A2-2 (aa 14-22)
TLLGWVNGL
A2-3 (7-15)
QILGVVLTL
CLDN6-specific T cell receptors
mC16#l:
SEQ ID NO: 6; > Va9N.3 J13 C
MLLALLSVLGIHFLLRDAQAQSVTQPDARVTVSEGASLQLRCKYSYFGTPYLFWYVQY
PRQGLQLLLKYYPGDPVVQGVNGFEAEFSKSNSSFHLRKASVHWSDWAVYFCAVSMSS
GTYQRFGTGTKLQVVPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQTNVPKTMESGTFI
TDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETD
MNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 7; > V629 D1 J2.5 C2
MRVRLISAVVLCFLGTGLVDMK‘.’TQMPRYLIKRMGENVLLECGQDMSHETM’YWYRQ
DPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSQNQ
DTQYFGPGTRLLVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW
VHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED
KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL
VLMAMVKKKNS
TCRCDs-mCl6#2:
SEQ ID NO: 8; > Va6N.6 J23 C
MDSFPGFVAVILLILGRTHGDSVTQTEGQVTVSESKSLIINCTYSATSIGYPNLFWYVRYP
GEGLQLLLKVITAGQKGSSRGFEATYNKEATSFHLQKASVQESDSAVYYCALNNQGKLI
FGQGTKLSIKPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVL
DMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQ
NLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 9; > V6132 D1 J2.4 C2
MGSRLFFVLSSLLCSKHMEAAVTQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDT
GHGLRLIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASGGDSQN
TLYFGAGTRLSVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWW
VNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDK
WPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL
MAMVKKKNS
TCRCDs—mCl6#3:
SEQ ID NO: 18; > Va16N J6 C
MLILSLLGAAFGSICFAATSMAQKVTQTQTSISVVEKTTVTMDCVYETRDSSYFLFWYK
QTASGEIVFLIRQDSYKKENATVGHYSLNFQKPKSSIGLIITATQIEDSAVYFCAMRDSSG
WO 50327 2015/056899
GNYKPTFGKGTSLVVHPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTF
ITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFET
DMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 19; > VB2 D2 .124 C2
MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAK
KPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQEDWG
SQNTLYFGAGTRLSVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELS
WWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSE
EDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVS
GLVLMAMVKKKNS
TCRCDs-mCl6#7:
SEQ ID NO: 28; > Va6N.7 0r Voc6D.7_4 J26 C
MDSFPGFMTVMLLIFTRAHGDSVTQTEGQVALSEEDFLTIHCNYSASGYPALFWYVQYP
GEGPQFLFRASRDKEKGSSRGFEATYDKGTTSFHLRKASVQESDSAVYYCALGNNYAQ
GLTFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDK
TVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNL
VMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 29; > VB13.3 D1 J1.4_02 C1
MGSRLFFV ’LILLCAKHMEAAVTQSPRSKVAVTGGKVTLSCHQTN‘NHDYMYWYRQDT
GHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSTGNERL
FFGHGTKLSVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVN
GKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWP
EGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM
AMVKRKNS
TCchs—mCl6#8:
SEQ ID NO:10;> V0.16N .113 C
MLILSLLGAAFGSICFATSMAQKVTQTQTSISVVEKTTVTMDCVYETRDSSYFLFWYKQ
TASGEIVFLIRQDSYKKENATVGHYSLNFQKPKSSIGLHTATQIEDSAVYFCAMREAANS
GTYQRFGTGTKLQVVPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFI
TDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETD
MNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID N0:11;> V132 D1 .113 C1
MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAK
KPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQQNSG
NTLYFGEGSRLIVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW
WVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED
KWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILY’EILLGKATLYAVLVSTLV
VMAMVKRKNS
TCRCDs-mCl6#10:
SEQ ID NO: 20; > Va13D.4_03 J42 C
MKRLVCSLLGLLCTQVCWVKGQQVQQSPASLVLQEGENAELQCNFSSTATRLQWFYQ
RPGGSLVSLLYNPSGTKHTGRLTSTTVTKERRSSLHISSSQTTDSGTYFCAMSSNSGGSN
AKLTFGKGTKLSVKSNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFIT
DKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETD
MNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 21; > VB4_02 D2 J2.7 C2
MGCRLLSCVAFCLLGIGPLETAVFQTPNYRVTRVGNEVSFNCEQTLDHNTMYWYKQDS
KKLLKIMFSYNNKQLIVNETVPRRFSPQSSDKAHLNLRIKSVELEDSAVYLCASSDWGDS
YEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW
VHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED
KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL
VLMAMVKKKNS
TCRCDs-mC16#12:
SEQ ID NO: 12; > Va3.3 J50 C
MKTVTGPLFLCFWLQLNCVSRGEQVEQRPPHLSVREGDSAVITCTYTDPNSYYFFWYK
QEPGASLQLLMKVFSSTEINEGQGFTVLLNKKDKRLSLNLTAAHPGDSAAYFCAVESSS
FSKLVFGQGTSLSVVPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFIT
DKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETD
MNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 13; > VBZ6 D2 .125 C2
MATRLLCYTVLCLLGARILNSKVIQTPRYLVKGQGQKAKMRCIPEKGHPVVFWYQQNK
NNEFKFLINFQNQEVLQQIDMTEKRFSAECPSNSPCSLEIQSSEAGDSALYLCASSLTGGA
QDTQYFGPGTRLLVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELS
EVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSE
EDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVS
GLVLMAMVKKKNS
TCchs-mCl6#13:
SEQ ID NO: 22; > Vu16N J22 C
MLILSLLGAAFGSICFAATSMAQKVTQTQTSISVVEKTTVTMDCVYETRDSSYFLFWYK
QTASGEIVFLIRQDSYKKENATVGHYSLNFQKPKSSIGLIITATQIEDSAVYFCAMRVASS
GSWQLIFGSGTQLTVMPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTF
ITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYP SSDVPCDATLTEKSFET
DMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 23; > VBZ D1 J2.1 C2
MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAK
FSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQGDNN
YAEQFFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELS
WWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSE
EDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVS
GLVLMAMVKKKNS
TCRcbs-mC16fll4:
SEQ ID NO: 14; > Va4N.4 or V014D.4w03 J6 C
AVLGILWVQICWVRGDQVEQSPSALSLHEGTGSALRCNFTTTMRAVQWFRK
NSRGSLINLFYLASGTKENGRLKSAFDSKERYSTLHIRDAQLEDSGTYFCAAEGGGNYK
PTFGKGTSLVVHPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKT
VLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNL
VMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID N0:15;>VB31D1J1.1C1
MLYSLLAFLLGMFLGVSAQTIHQWPVAEIKAVGSPLSLGCTIKGKSSPNLYWYWQATG
GTLQQLFYSITVGQVESVVQLNLSASRPKDDQFILSTEKLLLSHSGFYLCAWSPPINTEVF
FGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVN
GKE‘V’HSG‘v’STDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWP
EGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM
AMVKRKNS
TCchs—mCl6#15:
SEQ ID NO: 24; > Va3.1 J39 C
MKTVTGPLLLCFWLQLNCVSRGEQVEQRPPHLSVREGDSAIIICTYTDSATAYFSWYKQ
EAGAGLQLLMSVLSNVDRKEEQGLTVLLNKKDKRLSLNLTAAHPGDSAVYFCATNAG
AKLTFGGGTRLTVRPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFIT
DKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETD
NINLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ 11) N0: 25; > x434 D2 J2.7 C2
MGCRLLSCVAFCLLGIGPLETAVFQTPNYHVTQVGNEVSFNCKQTLGHDTMYWYKQD
SKKLLKIMFSYNNKQLIVNETVPRRFSPQSSDKAHLNLRIKSVEPEDSAVYLCASSLYWG
DSYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELS
WWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSE
EDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVS
GLVLMAMVKKKNS
TCRCDS-mCl6#17:
SEQ ID NO: 26; > Va14.3 or Val4D.3/DV8_08 J22 C
MDKNLTASFLLLGLHLAGVSGQQEKRDQQQVRQSPQSLTVWEGETAILNCSYENSAFD
YFPWYQQFPGEGPALLISILSVSDKKEDGRFTIFFNKREKKLSLHIADSQPGDSATYFCAA
SLSSGSWQLIFGSGTQLTVMPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTME
SGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEK
SFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 27; > VB?) D2 J2.7 C2
MDIWLLGWIIFSFLEAGHTGPKVLQIPSHQIIDMGQMVTLNCDPVSNHLYFYWYKQILG
QQMEFLVNFYNGKVMEKSKLFKDQFSVERPDGSYFTLKIQPTALEDSAVYFCASSLVGG
YEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW
VHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED
KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL
VLMAMVKKKNS
TCchs-mCl6#l8:
SEQ ID NO: 16; > Va6D.6_02 J4 C
MDSSPGFVAVILLILGRTHGDSVTQTEGPVTVSESESLIINCTYSATSIAYPNLFWYVRYP
GEGLQLLLKVITAGQKGSSRGFEATYNKETTSFHLQKASVQESDSAVYYCALGETGSFN
KLTFGAGTRLAVCP‘1’IQNPEPAV‘1’QLKDPRSQDSTLCLFTDFDSQI‘NVPKTMESGTFITD
KTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDM
NLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS
SEQ ID NO: 17; > V526 D1 J2.7 C2
MATRLLCYTVLCLLGARILNSKVIQTPRYLVKGQGQKAKMRCIPEKGHPVVFWYQQNK
NNEFKFLINFQNQEVLQQIDMTEKRFSAECPSNSPCSLEIQSSEAGDSALYLCASSLGIYE
QYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWV
SGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKW
PEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLM
AMVKKKNS
Claims (27)
1. A peptide comprising an amino acid sequence ed from the group consisting of SEQ ID NOs: 3, 4 and 5, wherein the peptide is 100 or less amino acids long.
2. The peptide of claim 1 which is 50 or less, 20 or less, or 10 or less amino acids long.
3. The peptide of claim 1 or 2 which consists of the amino acid sequence selected 10 from the group consisting of SEQ ID NOs: 3, 4 and 5.
4. A nucleic acid sing a nucleotide sequence encoding the peptide of any one of claims 1 to 3. 15
5. The nucleic acid of claim 4 which is a recombinant nucleic acid.
6. A cell comprising the nucleic acid of claim 4 or 5, wherein the cell is a not a cell in a human body. 20
7. A T cell receptor which binds to a x of the peptide of claim 3 with an MHC molecule.
8. An artificial T cell receptor which specifically binds to claudin-6 (CLDN6), sing a binding domain for CLDN6, a transmembrane domain and a T cell 25 signaling domain, wherein the binding domain for CLDN6 ses a single-chain variable fragment (scFv) of a CLDN6 antibody comprising a heavy chain variable region (VH) and a light chain le region (VL) which are covalently linked, wherein the binding domain for CLDN6 comprises a VH comprising the amino acid sequence according to SEQ ID NO: 32 and a VL comprising the amino acid sequence according 30 to SEQ ID NO: 39.
9. The cial T cell receptor of claim 8, wherein said heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected via a peptide linker. 5
10. The artificial T cell receptor of claim 9 wherein the e linker comprises the amino acid sequence (GGGGS)3.
11. The artificial T cell receptor of any one of claims 8 to 10, n the binding 10 domain for CLDN6 comprises the amino acid ce according to SEQ ID NO: 40.
12. The artificial T cell receptor of any one of claims 8 to 11, n the T cell ing domain comprises CD3-zeta. 15
13. The artificial T cell receptor of claim 12, wherein the signaling domain comprises an endodomain of CD3-zeta in combination with CD28.
14. The artificial T cell receptor of any one of claims 8 to 13 which comprises a signal peptide which directs the nascent protein into the endoplasmic reticulum.
15. The artificial T cell receptor of any one of claims 8 to 14 which comprises a spacer region which links the binding domain for CLDN6 to the transmembrane domain. 25
16. The artificial T cell receptor of any one of claims 8 to 15 which comprises the amino acid sequence according to SEQ ID NO: 46.
17. A nucleic acid comprising a nucleotide sequence encoding the T cell receptor of claim 7 or encoding the artificial T cell receptor of any one of claims 8 to 16.
18. A vector comprising nucleotide sequences encoding the T cell receptor of claim 7 or encoding the cial T cell receptor of any one of claims 8 to 16.
19. A composition comprising nucleotide sequences encoding the T cell receptor of claim 7 or encoding the artificial T cell receptor of any one of claims 8 to 16.
20. A cell comprising the T cell or of claim 7 or the artificial T cell receptor of 5 any one of claims 8 to 16 and/or comprising a nucleic acid comprising a nucleotide sequence encoding the T cell receptor chain or T cell receptor or encoding the artificial T cell receptor, wherein the cell is not a cell in a human body.
21. A method of producing an immunoreactive cell comprising the step of 10 transducing a T cell with a nucleic acid of claim 17, a vector of claim 18 or a ition of claim 19, n the T cell is not a cell in a human.
22. A ceutical ition comprising one or more of: (i) the peptide of any one of claims 1 to 3; 15 (ii) the nucleic acid of any one of claims 4, 5 and 17; (iii) the cell of claim 6 or 20; (iv) the T cell receptor of claim 7; and (v) the artificial T cell receptor of any one of claims 8 to 16. 20
23. The pharmaceutical composition of claim 22 which further comprises a pharmaceutically acceptable carrier.
24. Use of the pharmaceutical composition of claim 22 or 23 for the preparation of a medicament for treating or preventing a cancer e characterized by cancer cells 25 expressing CLDN6.
25. An ex vivo method for stimulating, priming and/or expanding T cells, comprising contacting T cells in vitro with one or more of: the peptide of any one of claims 1 to 3, the nucleic acid of claim 4 or 5 and/or the cell of claim 6.
26. A method for determining an immune response in a subject, comprising ining T cells reactive with a peptide of any one of claims 1 to 3 in a biological sample isolated from the subject, wherein the method comprises a step of contacting the sample with the peptide of any one of claims 1 to 3 and determining the level of T cell proliferation, wherein the observation of T cell proliferation tes that the sample comprises T cells reactive with the peptide. 5
27. Use of an agent for the preparation of a medicament for treating a cancer e by inducing a T cell response, wherein the cancer disease is characterized by cancer cells expressing CLDN6, wherein the agent is CLDN6 according to SEQ ID NO: 1 or 2 or a fragment thereof comprising the amino acid sequence according to SEQ ID NO: 3, 4 or 5, or a nucleic acid encoding CLDN6 or the nt thereof, or a cell presenting a 10 peptide consisting of the amino acid sequence according to SEQ ID NO: 3, 4 or 5 in the context of MHC.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP2014000868 | 2014-04-01 | ||
EPPCT/EP2014/000868 | 2014-04-01 | ||
EPPCT/EP2014/072864 | 2014-10-24 | ||
EP2014072864 | 2014-10-24 | ||
PCT/EP2015/056899 WO2015150327A1 (en) | 2014-04-01 | 2015-03-30 | Claudin-6-specific immunoreceptors and t cell epitopes |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ723544A NZ723544A (en) | 2021-10-29 |
NZ723544B2 true NZ723544B2 (en) | 2022-02-01 |
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