U.S. patent application number 16/595440 was filed with the patent office on 2020-01-30 for peptide mimotopes of claudin 18.2 and uses thereof.
The applicant listed for this patent is BioNTech AG, JPT Peptide Technologies GmbH, TRON - Translationale Onkologie an der Universitatsmedizin der Johannes Gutenberg-Universitat Mainz, Universitatsmedizin der Johannes Gutenberg-Universitat Mainz. Invention is credited to Matin Daneschdar, Markus Fiedler, Laura-Marie Kring (nee Plum), Ulf Reimer, Ugur Sahin, Hans-Ulrich Schmoldt, Karsten Schnatbaum.
Application Number | 20200031898 16/595440 |
Document ID | / |
Family ID | 50030242 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200031898 |
Kind Code |
A1 |
Sahin; Ugur ; et
al. |
January 30, 2020 |
PEPTIDE MIMOTOPES OF CLAUDIN 18.2 AND USES THEREOF
Abstract
The present invention provides molecules that mimic antigenic
determinants of the integral transmembrane protein claudin 18.2
(CLDN18.2). These molecules compete with CLDN18.2 for binding to a
CLDN18.2 binding domain, e.g. a CLDN18.2 binding domain of an
antibody, and are capable of detecting antibodies against CLDN18.2.
The mimotopes of the invention may be used to generate or inhibit
immune responses in animals and preferably humans. Furthermore,
they can be used for purposes of detecting agents comprising a
CLDN18.2 binding domain in biological samples as well as for
purifying agents comprising a CLDN18.2 binding domain.
Inventors: |
Sahin; Ugur; (Mainz, DE)
; Daneschdar; Matin; (Budenheim, DE) ; Schmoldt;
Hans-Ulrich; (Klein-Winternheim, DE) ; Kring (nee
Plum); Laura-Marie; (Mainz, DE) ; Fiedler;
Markus; (Halle an der Saale, DE) ; Reimer; Ulf;
(Berlin, DE) ; Schnatbaum; Karsten; (Berlin,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioNTech AG
TRON - Translationale Onkologie an der Universitatsmedizin der
Johannes Gutenberg-Universitat Mainz
Universitatsmedizin der Johannes Gutenberg-Universitat Mainz
JPT Peptide Technologies GmbH |
Mainz
Mainz
Mainz
Mainz |
|
DE
DE
DE
DE |
|
|
Family ID: |
50030242 |
Appl. No.: |
16/595440 |
Filed: |
October 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15113981 |
Jul 25, 2016 |
|
|
|
PCT/EP2014/000244 |
Jan 29, 2014 |
|
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16595440 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2500/02 20130101;
C07K 14/705 20130101; G01N 33/5308 20130101; G01N 2333/705
20130101; A61K 38/12 20130101; G01N 33/57492 20130101; A61K 38/08
20130101; C07K 7/64 20130101; G01N 2500/04 20130101; A61K 39/0005
20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; G01N 33/53 20060101 G01N033/53; A61K 39/00 20060101
A61K039/00; G01N 33/574 20060101 G01N033/574 |
Claims
1. A chimeric antigen receptor comprising a single-chain variable
fragment (scFV) fused to a CD3-zeta transmembrane and endodomain,
wherein said scFV comprises a heavy chain variable region (VH) and
a light chain variable region (VL), each comprising a set of three
complementarity-determining regions (CDR1, CDR2, and CDR3), wherein
the VH CDR1, CDR2, and CDR3 have the amino acid sequences of
positions 45-52, positions 70-77, and positions 116-126 of SEQ ID
NO: 12, respectively, and wherein the VL CDR1, CDR2, and CDR3 have
the amino acid sequences of positions 47-58, positions 76-78, and
positions 115-123 of SEQ ID NO: 13, respectively.
2. The chimeric antigen receptor of claim 1, wherein the scFV
further comprises a peptide linker connecting the VH and VL,
wherein the peptide linker comprises the amino acid sequence
(GGGGS)3, VE(GGSGGS)2GGVD, (GGGGS)4, (GGGGS)5, or
GGGGS(GGS)3GGGS.
3. The chimeric antigen receptor of claim 1, wherein the VH
comprises an amino acid sequence represented by SEQ ID NO: 2 and
the VL comprises an amino acid sequence represented by SEQ ID NO:
3.
4. The chimeric antigen receptor of claim 3, wherein the scFV
further comprises a peptide linker connecting the VH and VL,
wherein the peptide linker comprises the amino acid sequence
(GGGGS)3, VE(GGSGGS)2GGVD, (GGGGS)4, (GGGGS)5, or
GGGGS(GGS)3GGGS.
5. A recombinant nucleic acid encoding the chimeric antigen
receptor of claim 1.
6. The recombinant nucleic acid of claim 5, wherein the nucleic
acid is RNA.
7. A recombinant nucleic acid encoding the chimeric antigen
receptor of claim 3.
8. The recombinant nucleic acid of claim 7, wherein the nucleic
acid is RNA.
9. A cell comprising the chimeric antigen receptor of claim 1.
10. The cell of claim 9, wherein the cell is an immune effector
cell.
11. The cell of claim 9, wherein the cell is a T cell.
12. The cell of claim 11, wherein the chimeric antigen receptor is
on the surface of the T cell.
13. A cell comprising the chimeric antigen receptor of claim 3.
14. The cell of claim 13, wherein the cell is an immune effector
cell.
15. The cell of claim 13, wherein the cell is a T cell.
16. The cell of claim 15, wherein the chimeric antigen receptor is
on the surface of the T cell.
17. A method for treating a patient affected from a cancer
characterized by cancer cells expressing CLDN18.2 comprising a step
of administering to the patient the cell of claim 9.
18. A method for treating a patient affected from a cancer
characterized by cancer cells expressing CLDN18.2 comprising a step
of administering to the patient the cell of claim 11.
19. A method for treating a patient affected from a cancer
characterized by cancer cells expressing CLDN18.2 comprising a step
of administering to the patient the cell of claim 13.
20. A method for treating a patient affected from a cancer
characterized by cancer cells expressing CLDN18.2 comprising a step
of administering to the patient the cell of claim 15.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/113,981, which was a national stage entry
of international application PCT/EP2014/000244, filed on Jan. 29,
2014, the entire contents of which are incorporated herein by
reference.
[0002] The present invention provides molecules that mimic
antigenic determinants of the integral transmembrane protein
claudin 18.2 (CLDN18.2). These molecules compete with CLDN18.2 for
binding to a CLDN18.2 binding domain, e.g. a CLDN18.2 binding
domain of an antibody, and are capable of detecting antibodies
against CLDN18.2. The mimotopes of the invention may be used to
generate or inhibit immune responses in animals and preferably
humans. Additionally, they may serve as tools for the detection of
anti CLDN18.2 antibodies, CLDN18.2 binding domains or alternative
antibody formats in biological samples as well as for purification
purposes of said molecules.
[0003] Claudins 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 located in the cytoplasm. 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.
[0004] The claudin 18 (CLDN18) molecule is an integral
transmembrane protein (tetraspanin) having four membrane spanning
hydrophobic regions and two extracellular loops (loop1 embraced by
hydrophobic region 1 and hydrophobic region 2; loop2 embraced by
hydrophobic regions 3 and 4). CLDN18 exists in two different splice
variants, which are described in mouse and in human (Niimi, Mol.
Cell. Biol. 21:7380-90, 2001). The splice variants (Genbank
accession number: splice variant 1 (CLDN18.1): NP_057453,
NM_016369, and splice variant 2 (CLDN18.2): NM_001002026,
NP_001002026) have a molecular weight of approximately 27.9/27.72
kD. The splice variants CLDN18.1 and CLDN18.2 differ in the
N-terminal portion which comprises the first transmembrane (TM)
region and loop1, whereas the primary protein sequence of the
C-terminus is identical.
[0005] In normal tissues, there is no detectable expression of
CLDN18.2 with exception of stomach where CLDN18.2 is expressed
exclusively on short-lived differentiated gastric epithelial cells.
CLDN18.2 is maintained in the course of malignant transformation
and thus frequently displayed on the surface of human gastric
cancer cells. Moreover, this pan-tumoral antigen is ectopically
activated at significant levels in esophageal, pancreatic and lung
adenocarcinomas. The CLDN18.2 protein is also localized in lymph
node metastases of gastric cancer adenocarcinomas and in distant
metastases especially into the ovary (so-called Krukenberg
tumors).
[0006] IMAB362 is a highly tumor-specific monoclonal IgG1 antibody
currently in clinical development among others for the treatment of
advanced gastro-esophageal and stomach cancer [Sahin, U. et al.,
Clin. Cancer Res. 2008, 14, 7624-7634; Woll, S. et al., Int. J.
Cancer 2013, DOI: 10.1002/ijc.28400]. The antibody is directed
against the cancer specific cell surface target Claudin 18 isoform
2 (CLDN18.2). A phase I and a phase IIa trial have been conducted
in multiply pre-treated late-stage patients with advanced
gastro-esophageal cancer using IMAB362 as single agent and have
shown tolerability and antitumoral activity. In an on-going
randomized Phase IIb trial, IMAB362 is combined with standard
chemotherapy for first-line treatment of gastro-esophageal cancer
[Schuler, M. H. et al., J. Clin. Oncol. 2013, 31 (suppl; abstr
4080)].
[0007] For the development of IMAB362 towards clinical use the
detection and quantification of the antibody after application to
animals and patients is crucial for the characterization of ADME
and PK properties. Typically, ELISA-based assays are being applied
for this purpose using the corresponding antigen. Unfortunately,
transmembrane proteins are often difficult to produce and handle
due to their peculiar nature as membrane-embedded structures
[Scott, D. J. et al., Curr. Opin. Chem. Biol. 2013, 17, 427-435].
This obstacle can be overcome by using anti-idiotypic antibodies
which bind specifically to the antigen binding region of the
therapeutic antibody. However, generating such anti-idiotypic
antibodies is time-consuming and expensive. Therefore, mimotopes as
easier-to-prepare structures are considered useful substitutes to
mimic the full-length antigen, allowing tight specific binding of
the therapeutic antibody. Mimotopes can be proteins but also
oligopeptides [Casey, J. L. et al., J. Clin. Microbiol. 2006, 44,
764-771; Kieber-Emmons, T., Immunol. Res. 1998, 17, 95-108; Tang,
Y., et al., J. Biol. Chem. 1999, 274, 27371-27378; Wagner, S. et
al., Clin. Cancer Res. 2008, 14, 8178-8183].
[0008] As an alternative to protein ELISA, peptide ELISA is a
technique that is increasingly being used [Velumani, S. et al.,
PLoS. One 2011, DOI: 10.1371/journal.pone.0020737]. Peptides are
easily accessible by chemical synthesis, show higher stability and
are easier to handle compared to proteins. In general, peptide
ELISA enables the analysis of protein/protein interactions on the
amino acid sequence level, e.g. for definition of protein
interaction sites. Specifically binding peptides--or peptide-based
mimotopes--for peptide ELISA can be discovered with several
approaches which can be either knowledge-based or based on random
library approaches. Phage display is a well-established technique
for screening large protein or peptide libraries that often yield
strong binding partners for disease-related proteins or antibodies
[Molek, P. et al., Molecules 2011, 16, 857-887; Szardenings, M.,
Transduct. Res. 2003, 23, 307-349]. However, in some cases the
affinities or physicochemical properties of these hit peptides need
to be optimized. Here, peptide microarrays offer an efficient
approach. Thousands of peptides can be screened economically in
parallel requiring only small amounts of precious analyte. In
addition, assays on peptide microarrays can be set up rapidly
because the read-out is frequently achieved by standardized
fluorescence-based methods allowing low background when glass
surfaces are being used [Lorenz, P. et al., Methods Mol. Biol.
2009, 524, 247-258; Nahtman, T. et al., J. Immunol. Methods 2007,
328, 1-13; Thiele, A. et al., Methods Mol. Biol. 2009, 570, 19-65;
Pai, J. et al., J. Am. Chem. Soc. 2012, 134, 19287-19296].
[0009] It has been an object of the invention to prepare structures
which may serve as substitutes to mimic the full-length CLDN18.2
antigen and which are available in a format which is compatible
with biochemical formats for assay analytics and purification
procedures.
[0010] According to the invention, the systematic development of
peptide-based mimotopes for CLDN18.2 which bind to the drug
candidate IMAB362 is reported. The mimotope discovery and
optimization process was performed via a combined screening and
affinity maturation approach using phage display followed by a
peptide-microarray-based characterization of the
structure-activity-relationship (SAR) of the peptide hits leading
to rapid optimization of their binding properties. The resulting
mimotopes were shown to bind strongly and specifically and to be
well-suited for detection of the antibody in human and murine
serum.
[0011] The identification and isolation of molecules that mimic
antigenic determinants of CLDN18.2 provides significant advantages
and benefits. The use of molecules mimicking epitopes, and in
particular peptide based mimotopes, as diagnostic antigens is
advantageous because it allows focus on relevant single
specificities and avoids the diagnostically unimportant epitopes
present in complex antigens. Peptides of high quality and stability
can be cheaply and reproducibly produced and are easily applied to
ELISA as well as other formats.
SUMMARY OF THE INVENTION
[0012] The present invention provides a peptide mimotope of claudin
18.2 (CLDN18.2). In one embodiment, the peptide mimotope is a
structural mimic of either a linear or conformational CLDN18.2
epitope. In one embodiment, the peptide mimotope is a structural
mimic of an epitope in the extracellular domain of CLDN18.2.
[0013] In one embodiment, the peptide mimotope has a binding
capacity to a CLDN18.2 binding domain and/or competes with CLDN18.2
for binding to a CLDN18.2 binding domain. In one embodiment, the
CLDN18.2 binding domain is comprised by a binding agent to
CLDN18.2. In one embodiment, the binding agent to CLDN18.2 is
selected from the group consisting of an antibody or antibody
fragment to CLDN18.2, a bispecific or multispecific molecule
comprising a binding domain to CLDN18.2 and a chimeric antigen
receptor (CAR). In one embodiment, the binding agent to CLDN18.2 is
selected from the group consisting of artificial binding molecules
(scaffolds) including but not limited to nanobodies, affibodies,
anticalins, DARPins, monobodies, avimers, and microbodies. In one
embodiment, the bispecific molecule is a bispecific antibody. In
one embodiment, the bispecific antibody is a bispecific single
chain antibody. In one embodiment, the binding agent binds to an
epitope in the extracellular domain of CLDN18.2. In one embodiment,
said binding is a specific binding.
[0014] In one embodiment, the bispecific or multispecific molecule
comprises a first binding domain binding to CLDN18.2 and a second
binding domain binding to a T cell. In one embodiment, the
bispecific or multispecific molecule comprises a first binding
domain binding to CLDN18.2 and a second binding domain binding to
CD3. In one embodiment, said second binding domain binding to CD3
binds to the epsilon-chain of CD3. In one embodiment, said CD3 is
expressed on the surface of a T cell. In one embodiment, binding of
said binding agent to CD3 on T 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 apoptosis of cancer cells.
[0015] In one embodiment, the CLDN18.2 binding domain comprises a
variable domain of a heavy chain of an immunoglobulin (VH) with a
specificity for CLDN18.2 (VH(CLDN18.2)) and a variable domain of a
light chain of an immunoglobulin (VL) with a specificity for
CLDN18.2 (VH(CLDN18.2)). In one embodiment, said VH(CLDN18.2)
comprises an amino acid sequence represented by SEQ ID NO: 2 or a
fragment thereof or a variant of said amino acid sequence or
fragment and the VL(CLDN18.2) comprises an amino acid sequence
represented by SEQ ID NO: 3 or a fragment thereof or a variant of
said amino acid sequence or fragment.
[0016] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00001 (SEQ ID NO: 14) Xaa1 Xaa2 Xaa3 Tyr Xaa4 Xaa5
Xaa6
[0017] wherein
[0018] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of Gln,
His and Tyr,
[0019] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys and Tyr,
[0020] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Ala, Gly, Asn, Arg and Ser,
[0021] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Tyr, Pro and Arg, more preferably an
amino acid selected from the group consisting of Tyr and Pro,
[0022] Xaa5 is any amino acid, preferably an amino acid selected
from the group consisting of His, Gly, Lys and Arg, more preferably
an amino acid selected from the group consisting of His and Gly,
and
[0023] Xaa6 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg and Val.
[0024] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00002 (SEQ ID NO: 15) Cys Xaa1 Xaa2 Xaa3 Tyr Xaa4 Xaa5
Xaa6 Cys
[0025] wherein
[0026] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of Gln,
His and Tyr,
[0027] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys and Tyr,
[0028] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Ala, Gly, Asn, Arg and Ser,
[0029] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Tyr, Pro and Arg, more preferably an
amino acid selected from the group consisting of Tyr and Pro,
[0030] Xaa5 is any amino acid, preferably an amino acid selected
from the group consisting of His, Gly, Lys and Arg, more preferably
an amino acid selected from the group consisting of His and Gly,
and
[0031] Xaa6 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg and Val.
[0032] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00003 (SEQ ID NO: 16) Ala Cys Xaa1 Xaa2 Xaa3 Tyr Xaa4 Xaa5
Xaa6 Cys
[0033] wherein
[0034] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of Gln,
His and Tyr,
[0035] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys and Tyr,
[0036] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Ala, Gly, Asn, Arg and Ser,
[0037] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Tyr, Pro and Arg, more preferably an
amino acid selected from the group consisting of Tyr and Pro,
[0038] Xaa5 is any amino acid, preferably an amino acid selected
from the group consisting of His, Gly, Lys and Arg, more preferably
an amino acid selected from the group consisting of His and Gly,
and
[0039] Xaa6 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg and Val.
[0040] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00004 (SEQ ID NO: 17) Ala Cys Xaa1 Xaa2 Xaa3 Tyr Xaa4 Xaa5
Xaa6 Cys Gly
[0041] wherein
[0042] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of Gln,
His and Tyr,
[0043] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys and Tyr,
[0044] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Ala, Gly, Asn, Arg and Ser,
[0045] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Tyr, Pro and Arg, more preferably an
amino acid selected from the group consisting of Tyr and Pro,
[0046] Xaa5 is any amino acid, preferably an amino acid selected
from the group consisting of His, Gly, Lys and Arg, more preferably
an amino acid selected from the group consisting of His and Gly,
and
[0047] Xaa6 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg and Val.
[0048] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence Tyr Pro Gly.
[0049] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00005 (SEQ ID NO: 18) Xaa1 Xaa2 Xaa3 Tyr Pro Gly Xaa4
[0050] wherein
[0051] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of His
and Tyr,
[0052] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Leu,
Lys, Tyr and Phe, more preferably an amino acid selected from the
group consisting of Leu, Lys and Tyr,
[0053] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Gly, Asn, Arg, Ser, Trp and Lys, more preferably an
amino acid selected from the group consisting of Gly, Asn, Arg and
Ser, and
[0054] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Trp, Tyr, Glu, Arg, Val, Lys, Ile, Met
and Phe, more preferably an amino acid selected from the group
consisting of Trp, Tyr, Glu, Arg and Val.
[0055] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00006 (SEQ ID NO: 19) Cys Xaa1 Xaa2 Xaa3 Tyr Pro Gly Xaa4
Cys
[0056] wherein
[0057] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of His
and Tyr,
[0058] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Leu,
Lys, Tyr and Phe, more preferably an amino acid selected from the
group consisting of Leu, Lys and Tyr,
[0059] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Gly, Asn, Arg, Ser, Trp and Lys, more preferably an
amino acid selected from the group consisting of Gly, Asn, Arg and
Ser, and
[0060] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Trp, Tyr, Glu, Arg, Val, Lys, Ile, Met
and Phe, more preferably an amino acid selected from the group
consisting of Trp, Tyr, Glu, Arg and Val.
[0061] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00007 (SEQ ID NO: 20) Ala Cys Xaa1 Xaa2 Xaa3 Tyr Pro Gly
Xaa4 Cys
[0062] wherein
[0063] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of His
and Tyr,
[0064] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Leu,
Lys, Tyr and Phe, more preferably an amino acid selected from the
group consisting of Leu, Lys and Tyr,
[0065] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Gly, Asn, Arg, Ser, Trp and Lys, more preferably an
amino acid selected from the group consisting of Gly, Asn, Arg and
Ser, and
[0066] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Trp, Tyr, Glu, Arg, Val, Lys, Ile, Met
and Phe, more preferably an amino acid selected from the group
consisting of Trp, Tyr, Glu, Arg and Val.
[0067] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence
TABLE-US-00008 (SEQ ID NO: 21) Ala Cys Xaa1 Xaa2 Xaa3 Tyr Pro Gly
Xaa4 Cys Gly
[0068] wherein
[0069] Xaa1 is any amino acid, preferably an amino acid selected
from the group consisting of Gln, His, Tyr, Lys and Met, more
preferably an amino acid selected from the group consisting of His
and Tyr,
[0070] Xaa2 is any amino acid, preferably an amino acid selected
from the group consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more
preferably an amino acid selected from the group consisting of Leu,
Lys, Tyr and Phe, more preferably an amino acid selected from the
group consisting of Leu, Lys and Tyr,
[0071] Xaa3 is any amino acid, preferably an amino acid selected
from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys, Trp, Phe
and Tyr, more preferably an amino acid selected from the group
consisting of Gly, Asn, Arg, Ser, Trp and Lys, more preferably an
amino acid selected from the group consisting of Gly, Asn, Arg and
Ser, and
[0072] Xaa4 is any amino acid, preferably an amino acid selected
from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val, Ile,
Leu, Met, Ala, Phe and Lys, more preferably an amino acid selected
from the group consisting of Trp, Tyr, Glu, Arg, Val, Lys, Ile, Met
and Phe, more preferably an amino acid selected from the group
consisting of Trp, Tyr, Glu, Arg and Val.
[0073] In one embodiment, the peptide mimotope of the present
invention comprises an amino acid sequence selected from the group
consisting of:
TABLE-US-00009 (SEQ ID NO: 22) Gln Pro Ala Tyr Tyr His Thr, (SEQ ID
NO: 23) His Leu Gly Tyr Pro Gly Arg, (SEQ ID NO: 24) His Tyr Gly
Tyr Pro Gly Arg, (SEQ ID NO: 25) His Leu Gly Tyr Pro Gly Trp, (SEQ
ID NO: 26) His Tyr Ser Tyr Pro Gly Val, (SEQ ID NO: 27) His Tyr Gly
Tyr Pro Gly Val, (SEQ ID NO: 28) His Tyr Ser Tyr Pro Gly Trp, (SEQ
ID NO: 29) His Leu Arg Tyr Pro Gly Glu, (SEQ ID NO: 30) His Tyr Arg
Tyr Pro Gly Glu, (SEQ ID NO: 31) His Leu Asn Tyr Pro Gly Tyr, (SEQ
ID NO: 32) His Leu Gly Tyr Pro Gly Tyr, (SEQ ID NO: 33) His Leu Asn
Tyr Pro Gly Trp, (SEQ ID NO: 34) Tyr Lys Gly Tyr Pro Gly Tyr, (SEQ
ID NO: 35) His Tyr Gly Tyr Pro Gly Trp,
and
[0074] a fragment of said amino acid sequence or a variant of said
amino acid sequence or fragment.
[0075] In one embodiment, the peptide mimotope of the present
invention comprises an amino acid sequence selected from the group
consisting of:
TABLE-US-00010 (SEQ ID NO: 36) Cys Gln Pro Ala Tyr Tyr His Thr Cys,
(SEQ ID NO: 37) Cys His Leu Gly Tyr Pro Gly Arg Cys. (SEQ ID NO:
38) Cys His Tyr Gly Tyr Pro Gly Arg Cys, (SEQ ID NO: 39) Cys His
Leu Gly Tyr Pro Gly Trp Cys, (SEQ ID NO: 40) Cys His Tyr Ser Tyr
Pro Gly Val Cys, (SEQ ID NO: 41) Cys His Tyr Gly Tyr Pro Gly Val
Cys, (SEQ ID NO: 42) Cys His Tyr Ser Tyr Pro Gly Trp Cys, (SEQ ID
NO: 43) Cys His Leu Arg Tyr Pro Gly Glu Cys. (SEQ ID NO: 44) Cys
His Tyr Arg Tyr Pro Gly Glu Cys, (SEQ ID NO: 45) Cys His Leu Asn
Tyr Pro Gly Tyr Cys, (SEQ ID NO: 46) Cys His Leu Gly Tyr Pro Gly
Tyr Cys, (SEQ ID NO: 47) Cys His Leu Asn Tyr Pro Gly Trp Cys. (SEQ
ID NO: 48) Cys Tyr Lys Gly Tyr Pro Gly Tyr Cys, (SEQ ID NO: 49) Cys
His Tyr Gly Tyr Pro Gly Trp Cys,
and
[0076] a fragment of said amino acid sequence or a variant of said
amino acid sequence or fragment.
[0077] In one embodiment, the peptide mimotope of the present
invention comprises an amino acid sequence selected from the group
consisting of:
TABLE-US-00011 (SEQ ID NO: 50) Ala Cys Gln Pro Ala Tyr Tyr His Thr
Cys Gly, (SEQ ID NO: 51) Ala Cys His Leu Gly Tyr Pro Gly Arg Cys
Gly, (SEQ ID NO: 52) Ala Cys His Tyr Gly Tyr Pro Gly Arg Cys Gly,
(SEQ ID NO: 53) Ala Cys His Leu Gly Tyr Pro Gly Trp Cys Gly, (SEQ
ID NO: 54) Ala Cys His Tyr Ser Tyr Pro Gly Val Cys Gly, (SEQ ID NO:
55) Ala Cys His Tyr Gly Tyr Pro Gly Val Cys Gly, (SEQ ID NO: 56 Ala
Cys His Tyr Ser Tyr Pro Gly Trp Cys Gly, (SEQ ID NO: 57) Ala Cys
His Leu Arg Tyr Pro Gly Glu Cys Gly, (SEQ ID NO: 58) Ala Cys His
Tyr Arg Tyr Pro Gly Glu Cys Gly, (SEQ ID NO: 59) Ala Cys His Leu
Asn Tyr Pro Gly Tyr Cys Gly, (SEQ ID NO: 60) Ala Cys His Leu Gly
Tyr Pro Gly Tyr Cys Gly, (SEQ ID NO: 61) Ala Cys His Leu Asn Tyr
Pro Gly Trp Cys Gly, (SEQ ID NO: 62) Ala Cys Tyr Lys Gly Tyr Pro
Gly Tyr Cys Gly, (SEQ ID NO: 63) Ala Cys His Tyr Gly Tyr Pro Gly
Trp Cys Gly,
and
[0078] a fragment of said amino acid sequence or a variant of said
amino acid sequence or fragment.
[0079] In one embodiment, the peptide mimotope of the present
invention comprises the amino acid sequence His Pro Asp. In one
embodiment, the peptide mimotope of the present invention comprises
the amino acid sequence Tyr Leu His Pro Asp (SEQ ID NO: 64).
[0080] In one embodiment, the peptide mimotope of the present
invention comprises an amino acid sequence selected from the group
consisting of:
TABLE-US-00012 (SEQ ID NO: 65) Thr Pro Tyr His His Pro Asp Phe Pro
Tyr Trp Phe, (SEQ ID NO: 66) Tyr Leu His Pro Asp Tyr Pro, (SEQ ID
NO: 67) Tyr Leu His Pro Asp Val Met, (SEQ ID NO: 68) Pro Arg Cys
Lys Ser Glu Gly Pro His His Pro Asp Tyr Pro Asp Cys Arg Arg Asp Ser
Asp Cys Asn Gly Glu Cys Ile Cys Arg Gly Asn Gly Tyr Cys Gly, (SEQ
ID NO: 69) Ala Cys Arg His Pro Asp His Leu Asp Cys, (SEQ ID NO: 70)
Ala Cys His Glu Thr His His Pro Asp Cys,
and
[0081] a fragment of said amino acid sequence or a variant of said
amino acid sequence or fragment.
[0082] In other embodiments, the peptide mimotope of the present
invention comprises an amino acid sequence selected from the group
consisting of:
TABLE-US-00013 (SEQ ID NO: 71) Ser Phe Arg Asp Met Asn Tyr Ser Asp
Tyr Phe Met, (SEQ ID NO: 72) His Ile Leu Pro Leu Tyr Pro, (SEQ ID
NO: 73) Ser Pro Tyr Met Pro Met Gln, (SEQ ID NO: 74) Asp Arg Cys
Trp Leu Glu Gln Trp Pro Cys Arg Arg Asp Ser Asp Ile Pro, (SEQ ID
NO: 75) Gln Thr Cys Asp His Asp Thr Arg His Pro Thr Gly Asp Asp Leu
Cys Arg Arg Asp Ser Asp Cys Gly Gly Asn Cys Ile Cys Arg Gly Asn Gly
Tyr Cys Gly,
and
[0083] a fragment of said amino acid sequence or a variant of said
amino acid sequence or fragment.
[0084] In one embodiment, the peptide mimotope is conjugated to at
least one fusion partner. In one embodiment, the peptide mimotope
is part of a fusion polypeptide. In one embodiment, the fusion
partner comprises a heterologous amino acid sequence. In one
embodiment, the fusion partner comprises a reporter for an
immunological assay. In one embodiment, the reporter is selected
from the group consisting of alkaline phosphatase, horseradish
peroxidase, or a fluorescent molecule. In one embodiment, the
fusion partner comprises a label.
[0085] In one embodiment, the peptide mimotope is stabilized by a
covalent modification. Preferably, the modification is a
cyclization. In one embodiment, cyclization is via a disulfide, a
lactam, preferably a gamma-lactam, or another bridge.
[0086] In one embodiment, the peptide mimotopes and amino acid
sequences of peptide mimotopes described herein are comprised by a
structure conferring rigidity to the peptide mimotope or amino acid
sequence. For example, the peptide mimotope or amino acid sequence
may be inserted into a polypeptide or protein and may form a loop
of said polypeptide or protein. In one particularly preferred
embodiment, the peptide mimotopes described herein are cyclic
peptides and the amino acid sequences of peptide mimotopes
described herein are comprised by cyclic peptides.
[0087] In one embodiment, the peptide mimotope is present in
oligomeric or multimeric form. In this embodiment, two or more
peptide mimotopes of the invention which may be identical or
different may be linked or coupled by covalent or non-covalent
bonding, such as through biotin/streptavidin. Thus, peptide
mimotopes of the invention may form dimers, trimers, tetramers
etc.
[0088] The present invention further provides a recombinant nucleic
acid which encodes a peptide mimotope of the invention. In one
embodiment, the recombinant nucleic acid is in the form of a vector
or in the form of RNA.
[0089] The present invention further provides a host cell
comprising a recombinant nucleic acid of the invention.
[0090] The present invention further provides a method for assaying
for the presence and/or amount of CLDN18.2 in a sample comprising
using the peptide mimotope of the invention. In one embodiment, the
peptide mimotope is used as a competitor of the CLDN18.2, e.g. for
binding to an antibody against CLDN18.2
[0091] In particular, the present invention provides a method for
determining whether a sample contains CLDN18.2 which comprises
providing a monoclonal antibody against the CLDN18.2, reacting the
monoclonal antibody with the sample in a reaction mixture
containing the peptide mimotope of the invention as a competitor,
and determining whether the sample contains CLDN18.2.
[0092] The present invention also provides a method for determining
whether a sample contains CLDN18.2 which comprises: (a) incubating
in a reaction the sample, a monoclonal antibody against the
CLDN18.2, and a peptide mimotope of the invention which is a
competitor of the CLDN18.2 for the monoclonal antibody; (b)
detecting in the reaction a complex consisting of the CLDN18.2
bound by the monoclonal antibody and a complex formed by the
mimotope and monoclonal antibody; and (c) comparing an amount of
each of the complexes wherein a decrease in the amount of the
complex comprising the peptide mimotope indicates that the sample
contains CLDN18.2.
[0093] In one embodiment of the methods of the invention, the
peptide mimotope is conjugated to a label or a reporter.
[0094] The present invention further provides a method for assaying
for the presence and/or amount of binding agents to CLDN18.2 such
as CLDN18.2 antibodies in a sample comprising using the peptide
mimotope of the invention. Preferably, the peptide mimotope is used
for capturing binding agents to CLDN18.2 such as CLDN18.2
antibodies in a sample.
[0095] The present invention further provides a method for
capturing binding agents to CLDN18.2 such as CLDN18.2 antibodies in
a sample comprising using the peptide mimotope of the
invention.
[0096] In one embodiment, the methods of the invention are
performed in the context of an immunoassay.
[0097] In particular, the present invention provides a method of
determining CLDN18.2 antibodies in a sample which comprises
contacting a sample with at least one peptide mimotope of the
invention and assaying for the presence or absence of
mimotope-antibody complexes, wherein the presence of the
mimotope-antibody complexes is indicative of the presence of
CLDN18.2 antibodies in the sample.
[0098] In one embodiment of the methods of the invention, the
peptide mimotope is releasably or non-releasably immobilised on a
solid support.
[0099] The present invention also provides a method of separating
and/or purifying CLDN18.2 binding agents using the peptide mimotope
of the invention. In this embodiment, the peptide mimotope can be
used in the context of affinity chromatography. In particular, the
method may comprise contacting a sample with at least one peptide
mimotope of the invention and separating mimotope-CLDN18.2 binding
agent complexes from other components of the sample. In one
embodiment, the present invention provides a method of purifying a
CLDN18.2 binding agent, comprising treating a sample comprising the
CLDN18.2 binding agent with an immobilized peptide mimotope of the
invention, a washing step to separate off unwanted compounds such
as impurities, and an elution step to obtain the CLDN18.2 binding
agent.
[0100] The present invention further provides a test reagent or kit
comprising a peptide mimotope of the invention. In one embodiment,
the test reagent or kit is a diagnostic reagent or kit. In one
embodiment, the peptide mimotope is conjugated to a label.
According to the invention, a label may be selected from the group
consisting of a radioactive compound, a chemiluminescent compound,
an electroactive compound, a fluorescent compound, and a direct
particulate compound.
[0101] The test kit of the invention may further comprise at least
one additional reagent for performing an immunoassay and/or
instructions for use of the kit for performing an immunoassay.
[0102] The present invention further provides an assay device
comprising a peptide mimotope of the invention. In one embodiment,
the assay device is an enzyme-linked immunosorbent assay device. In
one embodiment, the peptide mimotope is releasably or
non-releasably immobilised on a solid support.
[0103] The present invention further provides a method for treating
a subject exposed to a CLDN18.2 binding agent, e.g. with the aim of
treating cancer, in particular cancer involving cells expressing
CLDN18.2, comprising treating the organism with a peptide mimotope
of the invention. In one embodiment, the peptide mimotope is
antagonistic to the CLDN18.2 binding agent. In one embodiment, the
peptide mimotope neutralizes binding of the CLDN18.2 binding agent
to CLDN18.2.
[0104] The present invention further provides the peptide mimotope
of the invention, the recombinant nucleic acid of the invention or
the host cell of the invention for use in therapy, in particular
for use in treating or preventing cancer in a patient, in
particular cancer involving cells expressing CLDN18.2.
[0105] The present invention further provides a pharmaceutical
composition comprising the peptide mimotope of the invention, the
recombinant nucleic acid of the invention or the host cell of the
invention.
[0106] The present invention further provides a method of treating
a patient, e.g. a patient having cancer, in particular cancer
involving cells expressing CLDN18.2, said patient preferably being
exposed to a CLDN18.2 binding agent, comprising administering to
the patient the pharmaceutical composition of the invention.
[0107] The present invention further provides a vaccine composition
comprising a peptide mimotope of the invention.
[0108] The present invention further provides a method for
eliciting antibodies against CLDN18.2 in a subject comprising
treating the subject with a peptide mimotope of the invention.
[0109] In one embodiment of all aspects of the invention, CLDN18.2
is expressed in a cancer cell, preferably on the surface of a
cancer cell.
[0110] According to the invention cancer cells expressing CLDN18.2
are preferably cancer cells of a cancer selected from the group
consisting of gastric cancer, esophageal cancer, pancreatic cancer,
lung cancer such as non small cell lung cancer (NSCLC), breast
cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck
cancer, cancer of the gallbladder and the metastasis thereof, a
Krukenberg tumor, peritoneal metastasis and/or lymph node
metastasis.
[0111] In one embodiment of all aspects of the invention, cancer is
preferably selected from the group consisting of gastric cancer,
esophageal cancer, pancreatic cancer, lung cancer such as non small
cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon
cancer, hepatic cancer, head-neck cancer, cancer of the gallbladder
and the metastasis thereof, a Krukenberg tumor, peritoneal
metastasis and/or lymph node metastasis.
[0112] In one embodiment of all aspects of the invention, CLDN18.2
preferably has the amino acid sequence according to SEQ ID NO:
1.
[0113] Other features and advantages of the instant invention will
be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0114] FIG. 1A and FIG. 1B. Single phage clone analysis. FIG. 1A
depicts phage ELISA binding analysis of 20 randomly picked clones
from the 3rd screening round. Rituximab, huIgG Fc fragment and the
secondary antibody ("2nd ab") were used to assess the specificity
of the phage presented peptides. HuIgG Fc: Fc fragment of human IgG
(Merck Millipore). 2nd ab: HRP conjugated goat anti-human IgG
antibody (Sigma). The mean of two experiments is shown. SD values
are presented as error bars. FIG. 1B depicts sequence alignment of
selected clones having a common `YPG` motif. The sequence alignment
of all IMAB362 binding clones is represented as WebLogo
(http://weblogo.berkeley.edu/).
[0115] FIG. 2. SAR analysis of IMAB362 binding peptides using
peptide microarrays. Shown are the results for substitutional
analyses of sequences number 1, 2 and 3. Each amino acid of the
starting sequence (shown at the bottom) is substituted by each of
the other natural amino acids except Cys, resulting in an overall
number of 7.times.19.times.3=399 different peptides. Dark colour
represents strong signals, shades show weaker signals. The blue
boxes indicate the amino acids of the original peptides. The
peptides highlighted with green boxes were selected for
re-synthesis, purification and detailed binding analysis.
[0116] FIGS. 3A, 3B, and 3C. Peptide ELISA with and without serum
incubation. FIG. 3A depicts comparison of the parental mimotope
peptide 2a with the corresponding maturated variants 2b and 2c in
the absence of serum. Control ab: antibody with identical
chimerized backbone compared to IMAB362, directed against a
different Claudin molecule. FIG. 3B depicts binding analysis of 2c
to IMAB362 in the presence of 0-20% mouse serum. FIG. 3C depicts
binding analysis of 2c to IMAB362 in the presence of 0-20% human
serum. Data were fitted with Sigma Plot using a two-site saturation
model.
[0117] FIG. 4. Pharmacokinetic analysis of IMAB362 using the
established peptide ELISA assay. IMAB362, Rituximab or PBS buffer
only were intravenously injected into Balb/-cJ mice. Blood samples
were taken over a period of 8 consecutive days and analyzed via
peptide ELISA with the 2c mimotope peptide for antibody capturing.
Median values of two mice per timepoint for each group are shown
and SD is presented as error bars for each timepoint.
[0118] FIG. 5. Biolayer interferometry of IMAB362 antibody
interaction with the mimotope peptides (1a, 1c, 2a, 2c, 3a, 3c).
Streptavidin biosensors were loaded with 5 .mu.g/ml of the
respective biotinylated peptide followed by incubation with
different IMAB362 concentrations (15.625, 31.25, 62.5, 125, 250,
500 nM). Shown are association and dissociation curves after
initial processing (subtraction of reference, Aligned to
baseline).
[0119] FIG. 6A and FIG. 6B. Effect of mimotopes on BiMAB-mediated
effects.
[0120] FIG. 6A depicts a bispecific single chain antibody specific
for CD3.epsilon. on T-cells and CLDN18.2 on cancer target cells was
pre-incubated with 20 .mu.g/ml CLDN18.2 mimotope complexed with
Neutravidin in a molar ratio of 1:8. Afterwards, the complex was
incubated with NugC4 target cells. In parallel, untreated BiMAB was
incubated with the cells as control. BiMAB binding to the
respective cells was detected by staining with an anti-His antibody
and an allophycocyanin-conjugated secondary antibody followed by
FACS analysis. Each sample was run in duplicates and SD is shown by
error bars.
[0121] FIG. 6B depicts NugC4 target cell lysis mediated by a
bispecific single chain antibody specific for CD3.epsilon. on
T-cells and CLDN18.2 on cancer target cells was assessed in a
cytotoxicity assay. Different concentrations of the BiMAB wer
pre-incubated with 20 .mu.g/ml of the CLDN18.2 mimotope complexed
with Neutravidin in a molar ratio of 1:8 before incubation in
presence of primary human T-cells and Luciferase-transfected NugC4
target cells for 24 hrs. Afterwards, luciferin was added to the
cells and luminescence was measured in a plate reader. To calculate
the specific lysis, minimum (NugC4 cells and T-cells) and maximum
(NugC4 cells and T-cells after addition of Triton-X-100 in a final
concentration of 2%) lysis was used. As a control the same assay
was performed without the addition of the mimotope complex. The
data shows mean values of triplicates with the corresponding SD
shown by error bars.
DETAILED DESCRIPTION OF THE INVENTION
[0122] Although the present invention is described in detail below,
it is to be understood 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 scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art.
[0123] 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 embodiments 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 embodiments 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 otherwise.
[0124] Preferably, the terms used herein are defined as described
in "A multilingual glossary of biotechnological terms: (IUPAC
Recommendations)", H. G. W. Leuenberger, B. Nagel, and H. Kolbl,
Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland,
(1995).
[0125] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, cell biology, immunology, and recombinant DNA
techniques which are explained in the literature in the field (cf.,
e.g., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, J.
Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor 1989).
[0126] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated member, integer or step or group
of members, integers or steps but not the exclusion 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 member,
integer or step or group of members, 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 otherwise indicated
herein, each individual value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(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.
[0127] 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 infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0128] Claudins are a family of proteins that are the most
important components of tight junctions, 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 spanning the membrane 4 times with the
N-terminal and the C-terminal end both located in the cytoplasm.
The first extracellular loop, termed EC1 or ECL1, consists on
average of 53 amino acids, and the second extracellular loop,
termed EC2 or ECL2, consists of around 24 amino acids. Cell surface
proteins of the claudin family, such as CLDN18.2, are expressed in
tumors of various origins, and are particularly suited as target
structures in connection with antibody-mediated cancer
immunotherapy due to their selective expression (no expression in a
toxicity relevant normal tissue) and localization to the plasma
membrane.
[0129] The term "CLDN" as used herein means claudin and includes
CLDN18.2. Preferably, a claudin is a human claudin.
[0130] The term "CLDN18" relates to claudin 18 and includes any
variants, including claudin 18 splice variant 1 (claudin 18.1
(CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2
(CLDN18.2)).
[0131] The term "CLDN18.2" preferably relates to human CLDN18.2,
and, in particular, to a protein comprising, preferably consisting
of the amino acid sequence according to SEQ ID NO: 1 of the
sequence listing or a variant of said amino acid sequence. The
first extracellular loop of CLDN18.2 preferably comprises amino
acids 27 to 81, more preferably amino acids 29 to 78 of the amino
acid sequence shown in SEQ ID NO: 1. The second extracellular loop
of CLDN18.2 preferably comprises amino acids 140 to 180 of the
amino acid sequence shown in SEQ ID NO: 1. Said first and second
extracellular loops preferably form the extracellular portion of
CLDN18.2.
[0132] CLDN18.2 is selectively expressed in normal tissues in
differentiated epithelial cells of the gastric mucosa. CLDN18.2 is
expressed in cancers of various origins such as pancreatic
carcinoma, esophageal carcinoma, gastric carcinoma, bronchial
carcinoma, breast carcinoma, and ENT tumors. CLDN18.2 is a valuable
target for the prevention and/or treatment of primary tumors, such
as gastric cancer, esophageal cancer, pancreatic cancer, lung
cancer such as non small cell lung cancer (NSCLC), ovarian cancer,
colon cancer, hepatic cancer, head-neck cancer, and cancers of the
gallbladder, and metastases thereof, in particular gastric cancer
metastasis such as Krukenberg tumors, peritoneal metastasis, and
lymph node metastasis.
[0133] The term "variant" according to the invention refers, in
particular, to mutants, splice variants, conformations, isoforms,
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
posttranslationally modified variants and conformation
variants.
[0134] According to the invention, the term "claudin positive
cancer" or similar terms means a cancer involving cancer cells
expressing a claudin, preferably on the surface of said cancer
cells.
[0135] "Cell surface" is used in accordance with its normal meaning
in the art, and thus includes the outside of the cell which is
accessible to binding by proteins and other molecules
[0136] A claudin is expressed on the surface of cells if it is
located at the surface of said cells and is accessible to binding
by claudin-specific antibodies added to the cells.
[0137] The term "extracellular portion" in the context of the
present invention refers to a part of a molecule such as a protein
that is facing the extracellular 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.
[0138] The terms "part" or "fragment" are used interchangeably
herein and refer to a continuous element. 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 or peptide is preferably immunologically
equivalent to the epitope or peptide it is derived from. A part or
fragment of a protein sequence preferably comprises a sequence of
at least 6, in particular 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.
[0139] According to the invention, CLDN18.2 is not substantially
expressed in a cell if the level of expression is lower compared to
expression in stomach cells or stomach 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 stomach cells or
stomach tissue or even lower. Preferably, CLDN18.2 is not
substantially expressed in a cell if the level of expression
exceeds the level of expression in non-cancerous tissue other than
stomach by no more than 2-fold, preferably 1.5-fold, and preferably
does not exceed the level of expression in said non-cancerous
tissue. Preferably, CLDN18.2 is not substantially expressed in a
cell if the level of expression is below the detection limit and/or
if the level of expression is too low to allow binding by
CLDN18.2-specific antibodies added to the cells.
[0140] According to the invention, CLDN18.2 is expressed in a cell
if the level of expression exceeds the level of expression in
non-cancerous tissue other than stomach preferably by more than
2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold.
Preferably, CLDN18.2 is expressed in a cell if the level of
expression is above the detection limit and/or if the level of
expression is high enough to allow binding by CLDN18.2-specific
antibodies added to the cells. Preferably, CLDN18.2 expressed in a
cell is expressed or exposed on the surface of said cell.
[0141] According to the present invention the term "mimotope"
refers to a molecule which is a mimic of an epitope. The mimotope
may also act as a competitor for the epitope of which it is a mimic
in in vitro assays (e.g. ELISA assays) and preferably binds to the
same antigen-binding region of an antibody which binds
immunospecifically to an epitope of a desired antigen. The mimotope
may elicit an immunological response in a host that is reactive to
the antigen of which it is a mimic.
[0142] According to the invention, peptide mimotopes can be
synthetically produced by chemical synthesis methods which are well
known in the art, either as an isolated peptide or as a part of
another peptide or polypeptide. Alternatively, a peptide mimotope
can be produced in a microorganism which produces the peptide
mimotope which is then isolated and if desired, further purified.
Thus, the peptide mimotope can be produced in microorganisms such
as bacteria, yeast, or fungi; in a eukaryote cells such as
mammalian or insect cells; or, in a recombinant virus vector such
as adenovirus, poxvirus, herpesvirus, Simliki forest virus,
baculovirus, bacteriophage, sindbis virus, or sendai virus.
Suitable bacteria for producing the peptide mimotope include
Escherichia coli, Bacillus subtilis, or any other bacterium that is
capable of expressing peptides such as the peptide mimotope.
Suitable yeast types for expressing the peptide mimotope include,
but are not limited to Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Candida, or any other yeast capable of
expressing peptides. Methods for using the aforementioned bacteria,
recombinant virus vectors, eukaryote cells to produce peptides are
well known in the art.
[0143] To produce the peptide mimotope, the nucleic acid encoding
the peptide mimotope is preferably in a plasmid and the nucleic
acid is operably linked to a promoter which effects expression of
the peptide mimotope in a microorganism. Suitable promoters
include, but are not limited to, T7 phage promoter, T3 phage
promoter, .beta.-galactosidase promoter, and the Sp6 phage
promoter. Methods for isolating and purifying peptides are well
known in the art and include methods such as gel filtration,
affinity chromatography, ion exchange chromatography, or
centrifugation.
[0144] The peptide mimotopes, either by themselves or as part of a
fusion peptide, can be conjugated to a heterologous peptide or
protein. Such heterologous proteins include, but are not limited
to, carrier proteins such as bovine serum albumen (BSA), and
reporter enzymes which include, but are not limited to, horseradish
peroxidase or alkaline phosphatase. Further, the peptide mimotopes
or fusion peptides comprising the peptide mimotope can be
chemically conjugated to fluorescent reporter molecules which
include, but are not limited to, fluorescein or R-phycoerythrin.
Methods for conjugating carrier proteins, enzymes, and fluorescent
reporter molecules to peptides and fusion peptides are well known
in the art.
[0145] To facilitate isolation of the peptide mimotope, a fusion
polypeptide can be made wherein the peptide mimotope is
translationally fused (covalently linked) to a heterologous tag
such as a heterologous polypeptide or polyhistidine, preferably six
histidine residues, which allows for the simplified recovery of the
fusion polypeptide, e.g. its isolation by affinity chromatography
or metal affinity chromatography, preferably nickel affinity
chromatography. In some instances it can be desirable to remove the
tag after purification. Therefore, it is also contemplated that the
fusion polypeptide comprises a cleavage site at the junction
between the peptide mimotope and the heterologous tag. The cleavage
site consists of an amino acid sequence that is cleaved with an
enzyme specific for the amino acid sequence at the site.
[0146] The peptide mimotopes described herein can be used as a
control or competitor in immunoassays for detecting CLDN18.2 and
can be used for detecting CLDN18.2 binding molecules such as
CLDN18.2 antibodies. The peptide mimotopes can further be used in
therapies for treating animals or humans exposed to CLDN18.2
binding molecules such as therapeutic CLDN18.2 antibodies, e.g. for
modulating, in particular reducing, the activity of the CLDN18.2
binding molecule.
[0147] For example, the peptide mimotopes, either alone, or as a
component of a fusion polypeptide, such as conjugated to a carrier
protein or fluorescent reporter molecule, are useful as standard
and conjugates in immunoassays such as ELISAs and RIAS, which are
used to determine whether a sample contains CLDN18.2. In such
immunoassays, the use of CLDN18.2 as a control or as a competitor
has been difficult. Therefore, the peptide mimotopes provide a
significant advantage over CLDN18.2.
[0148] In general, the immunoassays are performed using an
enzyme-linked immunosorbent assay (ELISA) embodiment.
[0149] A microtiter plate may be provided containing a plurality of
wells wherein a first well or series of wells contains a monoclonal
antibody against CLDN18.2 immobilized to the surface therein. A
sample may be mixed with the peptide mimotope and the mixture added
to the wells containing the bound monoclonal antibody. The mimotope
peptide may be part of a fusion polypeptide. The CLDN18.2 in the
sample and the peptide mimotope compete for binding to the
monoclonal antibody. The ELISA is incubated for a time sufficient
for antibody complexes to form. Afterwards, the wells are washed to
remove any unbound material. The wells may then be incubated with a
labeled antibody or an antibody conjugated to a reporter molecule
that binds to the fusion polypeptide to form a complex which can be
detected. A detectable signal from the reporter may indicate that
the sample does not contain CLDN18.2 whereas an absence of a signal
may indicate that the sample contains CLDN18.2 which had bound all
of the monoclonal antibody, thereby preventing the peptide mimotope
from binding the monoclonal antibody immobilized in the wells. When
the fusion polypeptide comprises a label or reporter molecule such
as a reporter enzyme such as alkaline phosphatase, the
antibody-mimotope peptide complex can be detected directly without
the need for a labeled antibody.
[0150] Alternatively, a microtiter plate may be provided containing
a plurality of wells wherein a first well or series of wells
contains the peptide mimotope, which may be conjugated to a carrier
protein or fusion polypeptide, immobilized to the surface therein.
Sample may be added to the wells containing the bound peptide
mimotopes along with a constant amount of a monoclonal antibody
against CLDN18.2. The CLDN18.2 in the sample and the peptide
mimotope bound to the well surfaces compete for binding to the
monoclonal antibody. The ELISA is incubated for a time sufficient
for antibody complexes to form. Afterwards, the wells are washed to
remove any unbound material. The amount of monoclonal antibody that
is bound to the immobilized mimotope peptides in the well is
determined by incubating the wells with a labeled antibody or an
antibody conjugated to a reporter molecule that binds to the
antibody against CLDN18.2 to form a complex that can be detected. A
detectable signal from the reporter indicates the sample does not
contain CLDN18.2 whereas an absence of a signal indicates that the
sample contains CLDN18.2 which had bound all of the antibody
against CLDN18.2, thereby preventing the antibody from binding the
peptide mimotope immobilized in the wells. The intensity of the
signal provides an estimate of the relative concentration of
CLDN18.2 in the sample. Alternatively, the antibody against
CLDN18.2 can be labeled with a reporter in which case the bound
antibody can be detected directly without the need for a labeled
antibody. In either case, detection is by methods well known in the
art for detecting the particular reporter ligand.
[0151] Instead of an ELISA, the peptide mimotopes can be used in a
radio immunoassay (RIA) for detecting CLDN18.2 in a sample. The RIA
procedure may involve incubation of a monoclonal antibody against
CLDN18.2, simultaneously with a solution of unknown sample or known
standard, and a constant amount of radioactively labeled peptide
mimotope or fusion polypeptide. After separation of the free
peptide mimotope or fusion polypeptide from bound peptide mimotope
or fusion polypeptide, the radioactivity in the respective
fractions is determined. The concentration of CLDN18.2 in the
unknown sample is determined by comparing results to a standard
curve. Several known methods may be used for the separation of free
from bound peptide mimotope or fusion polypeptide in the RIA.
Radioactivity may be determined in a liquid scintillation
counter.
[0152] According to the invention, the CLDN18.2 which is to be
assayed may be expressed on the surface of a cell.
[0153] Mimotopes of the invention may also be used in methods for
detecting the presence of antibodies against CLDN18.2. The design
of suitable immunoassays to put these methods into effect may be
subject to a great deal of variation, and a variety of these
immunoassays are known in the art. Suitable immunoassay protocols
may be based, for example, upon competition, or direct reaction, or
sandwich type assays. The immunoassay protocols used may also, for
example, use solid supports, or may be by immunoprecipitation.
Assays may involve the use of labelled antibodies and the labels
may be, for example, fluorescent, chemiluminescent, radioactive, or
dye molecules. Particular preferred assays are enzyme-labelled and
mediated immunoassays, such as ELISA assays.
[0154] Accordingly, the peptide mimotopes may also be used in an
assay such as an ELISA assay to determine antibody against CLDN18.2
in a sample. For this purpose, the wells of ELISA plates may be
coated with peptide mimotopes. Subsequently, a sample such as
plasma may be added and the detection of peptide specific
antibodies (primary antibody) may be performed with a labelled
secondary antibody directed against the primary antibody.
[0155] Mimotopes of the invention may be bound to a solid support,
for example the surface of an immunoassay well or dipstick, and/or
packaged into kits in a suitable container along with suitable
reagents, controls, instructions and the like.
[0156] Accordingly the present invention also provides a kit
comprising at least one mimotope of the present invention. In a
preferred embodiment, the kit further comprises at least one
additional agent such as one or more suitable reagents for
performing an immunoassay, a control, or instructions for use of
the kit.
[0157] When used as an assay reagent as described herein, a
mimotope of the invention may be conjugated to a label. Preferably,
the label is any entity the presence of which can be readily
detected. Preferably the label is a direct label. Direct labels are
entities which, in their natural state, are readily visible either
to the naked eye, or with the aid of an optical filter and/or
applied stimulation, e.g. UV light to promote fluorescence.
Examples include radioactive, chemiluminescent, electroactive (such
as redox labels), and fluorescent compounds. Direct particulate
labels, such as dye sols, metallic sols (e.g. gold) and coloured
latex particles, are also very suitable and are, along with
fluorescent compounds, preferred. Of these options, coloured latex
particles and fluorescent compounds are most preferred.
Concentration of the label into a small zone or volume should give
rise to a readily detectable signal, e.g. a strongly coloured area.
Indirect labels, such as enzymes, e.g. alkaline phosphatase and
horseradish peroxidase, can also be used, although these usually
require the addition of one or more developing reagents such as
substrates before a visible signal can be detected.
[0158] Conjugation of the label to the mimotope of the invention
can be by covalent or non-covalent (including hydrophobic) bonding,
or by adsorption. Techniques for such conjugation are commonplace
in the art and may be readily adapted for the particular reagents
employed.
[0159] According to the invention there is further provided an
assay device comprising at least one mimotope of the present
invention. In one embodiment, the assay device is selected from the
group consisting of an enzyme-linked immunosorbent assay
device.
[0160] Such a device can take different forms, and it can be varied
depending on the precise nature of the assay being performed. For
example, the mimotope of the invention may be coated onto a solid
support, typically nitrocellulose or other hydrophobic porous
material. Alternatively, the mimotope may be coated on a synthetic
plastics material, microtitre assay plate, microarray chip, latex
bead, filter comprising a cellulosic or synthetic polymeric
material, glass or plastic slide, dipstick, capillary fill device
and the like. Coating of the mimotopes to these surfaces can be
accomplished by methods known in the art. Protein carriers are
typically used for complexing, with BSA or adhesive peptides being
the most preferred. In one embodiment, the mimotope of the
invention is releasably immobilised on the solid support. In a
further preferred embodiment, the diagnostic reagent is
nonreleasably immobilised on the solid support.
[0161] The mimotopes of the invention may be further used as
vaccines so as to induce CLDN18.2 antibodies or for modulating the
activity of CLDN18.2 binding agents such as CLDN18.2 antibodies, in
particular bispecific antibodies binding to CLDN18.2. To this end,
the mimotopes of the invention may be combined with various
components to produce pharmaceutically acceptable compositions.
[0162] 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 specific symptoms and signs. A
disease may be caused by factors originally from an external
source, 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 problems 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 usually affect
individuals not only physically, but also emotionally, as
contracting and living with many diseases can alter one's
perspective on life, and one's personality. According to the
invention, the term "disease" includes cancer, in particular those
forms of cancer described herein. 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 CLDN18.2.
[0163] "Diseases involving cells expressing CLDN18.2" or similar
expressions means according to the invention that CLDN18.2 is
expressed in cells of a diseased tissue or organ. In one
embodiment, expression of CLDN18.2 in cells of a diseased 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
particular at least 20%, 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 CLDN18.2 include
cancer diseases. Furthermore, according to the invention, cancer
diseases preferably are those wherein the cancer cells express
CLDN18.2.
[0164] The terms "cancer disease" or "cancer" refer to or describe
the physiological condition in an individual that is typically
characterized by unregulated cell growth. Examples of cancers
include, but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particularly, examples of such cancers
include bone cancer, blood cancer, 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, prostate cancer, uterine cancer, carcinoma
of the sexual and reproductive organs, Hodgkin's Disease, cancer of
the esophagus, cancer of the small intestine, cancer of the
endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma 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), neuroectodermal cancer, spinal axis tumors,
glioma, meningioma, and pituitary adenoma. The term "cancer"
according to the invention also comprises cancer metastases.
Preferably, a "cancer disease" is characterized by cells expressing
CLDN18.2 and a cancer cell expresses CLDN18.2. A cell expressing
CLDN18.2 preferably is a cancer cell, preferably of the cancers
described herein.
[0165] 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
malignant cells from the primary tumor, invasion 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 depends 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 metastasis which is remote from the
primary tumor and the regional lymph node system. In one
embodiment, the term "metastasis" according to the invention
relates to lymph node metastasis. One particular form of metastasis
which is treatable using the therapy of the invention is metastasis
originating from gastric cancer as primary site. In preferred
embodiments such gastric cancer metastasis is Krukenberg tumors,
peritoneal metastasis and/or lymph node metastasis.
[0166] Krukenberg tumor is an uncommon metastatic tumor of the
ovary accounting for 1% to 2% of all ovarian tumors. Prognosis of
Krukenberg tumor is still very poor and there is no established
treatment for Krukenberg tumors. Krukenberg tumor is a metastatic
signet ring cell adenocarcinoma of the ovary. Stomach is the
primary site in most Krukenberg tumor cases (70%). Carcinomas of
colon, appendix, and breast (mainly invasive lobular carcinoma) are
the next most common primary sites. Rare cases of Krukenberg tumor
originating from carcinomas of the gallbladder, biliary tract,
pancreas, small intestine, ampulla of Vater, cervix, and urinary
bladder/urachus have been reported.
[0167] 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 disease in an individual, arrest or slow the
development of a disease in an individual, inhibit or slow the
development of a disease in an individual, decrease the frequency
or severity of symptoms in an individual, and/or decrease the
recurrence in an individual who currently has or who previously has
had a disease.
[0168] The terms "prophylactic treatment" or "preventive treatment"
relate to any treatment that is intended to prevent a disease from
occurring in an individual. The terms "prophylactic treatment" or
"preventive treatment" are used herein interchangeably.
[0169] The terms "immunization" or "vaccination" describe the
process of administering an antigen to an individual with the
purpose of inducing an immune response, for example, for
therapeutic or prophylactic reasons.
[0170] The terms "protect", "prevent", "prophylactic",
"preventive", or "protective" relate to the prevention and/or
treatment of the occurrence and/or the propagation of a disease,
e.g. tumor, in an individual. For example, a prophylactic
administration of a therapy can protect the receiving individual
from the development of a disease.
[0171] The terms "individual" and "subject" are used herein
interchangeably. They refer to human beings, non-human primates or
other mammals (e.g. mouse, rat, rabbit, dog, cat, cattle, swine,
sheep, horse or primate) that can be afflicted with or are
susceptible to a disease or disorder (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
"individual" and "subject" do not denote a particular age, and thus
encompass adults, elderlies, children, 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 subject.
[0172] "Target cell" shall mean any undesirable cell such as a
cancer cell. In preferred embodiments, the target cell expresses
CLDN18.2.
[0173] The term "antigen" relates to an agent comprising an epitope
against which an immune response is to be generated and/or is
directed. The term "antigen" includes in particular proteins,
peptides, polysaccharides, nucleic acids, especially RNA and DNA,
and nucleotides. The term "antigen" also includes agents, which
become antigenic--and sensitizing--only through transformation
(e.g. intermediately in the molecule or by completion with body
protein). An antigen or a processing product thereof is preferably
recognizable by a T or B cell receptor, or by an immunoglobulin
molecule such as an antibody. In a preferred embodiment, the
antigen is a disease-associated antigen, such as a tumor antigen, a
viral antigen, or a bacterial antigen.
[0174] In the context of the present invention, the term "tumor
antigen" or "tumor-associated antigen" preferably relates to
proteins that are under normal conditions specifically expressed in
a limited number of tissues and/or organs or in specific
developmental stages and are expressed or aberrantly expressed in
one or more tumor or cancer tissues. 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. For example, CLDN18.2 has
been identified as differentially expressed in tumor tissues, with
the only normal tissues expressing CLDN18.2 being stomach
[0175] The term "epitope" refers to an antigenic determinant in a
molecule, i.e., to the part in a molecule that is recognized by the
immune system, for example, that is recognized by an antibody. For
example, epitopes are the discrete, three-dimensional sites on an
antigen, which are recognized by the immune system. Epitopes
usually consist of chemically active surface groupings of molecules
such as amino acids or sugar side chains and usually have specific
three dimensional structural characteristics, as well as specific
charge characteristics. Conformational and non-conformational
epitopes are distinguished in that the binding to the former but
not the latter is lost in the presence of denaturing solvents. An
epitope of a protein 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 preferably between 10 and 25 amino acids in length, for
example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in
length.
[0176] According to the invention, the term "binding agent to
CLDN18.2" includes any compound that has a binding capacity to
CLDN18.2. Preferably, such binding agent comprises at least one
binding domain for CLDN18.2. The term includes molecules such as
antibodies and antibody fragments, bispecific or multispecific
molecules and chimeric antigen receptors (CARs). The term also
includes all artificial binding molecules (scaffolds) having a
binding capacity to CLDN18.2 including but not limited to
nanobodies, affibodies, anticalins, DARPins, monobodies, avimers,
and microbodies. In one embodiment said binding agent binds to an
extracellular domain of CLDN18.2. In one embodiment said binding
agent binds to native epitopes of CLDN18.2 present on the surface
of living cells. In one embodiment said binding agent binds to the
first extracellular loop of CLDN. In one embodiment said binding to
CLDN18.2 is a specific binding.
[0177] In one embodiment the binding domain binding to CLDN18.2
comprises a variable domain of a heavy chain of an immunoglobulin
(VH) with a specificity for a claudin 18.2 antigen (VH(CLDN18.2))
and a variable domain of a light chain of an immunoglobulin (VL)
with a specificity for a claudin 18.2 antigen (VL(CLDN18.2)).
[0178] In one embodiment said VH(CLDN18.2) comprises an amino acid
sequence represented by SEQ ID NO: 2 or a fragment thereof or a
variant of said amino acid sequence or fragment and the
VL(CLDN18.2) comprises an amino acid sequence represented by SEQ ID
NO: 3 or a fragment thereof or a variant of said amino acid
sequence or fragment.
[0179] 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
conserved, 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 following order: FR1, CDR1, FR2, 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
(C1q) of the classical complement system.
[0180] The term "monoclonal antibody" as used herein refers to a
preparation of antibody molecules of single molecular composition.
A monoclonal antibody displays a single binding specificity and
affinity. In one embodiment, the monoclonal 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.
[0181] The term "recombinant antibody", as used herein, includes
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, (c) antibodies isolated from a
recombinant, combinatorial antibody library, and (d) antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of immunoglobulin gene sequences to other DNA
sequences.
[0182] 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
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in
vivo).
[0183] The term "humanized antibody" refers to a molecule having an
antigen binding site that is substantially derived from an
immunoglobulin 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 complementarity determining
regions (CDR) grafted onto appropriate framework regions 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 antibodies 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.
[0184] 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 presently
known sources using readily available B-cells or hybridomas from
non-human host organisms in combination with constant regions
derived from, for example, human cell preparations. While the
variable region has the advantage of ease of preparation and the
specificity is not affected by the source, the constant region
being human, is less likely to elicit an immune response from a
human subject when the antibodies are injected than would the
constant region from a non human source. However the definition is
not limited to this particular example.
[0185] Antibodies may be derived from different species, including
but not limited to mouse, rat, rabbit, guinea pig and human.
[0186] Antibodies described herein include IgA such as IgA1 or
IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In
various embodiments, the antibody is an IgG1 antibody, more
particularly an IgG1, kappa or IgG1, lambda isotype (i.e. IgG1,
.kappa., .lamda.), an IgG2a antibody (e.g. IgG2a, .kappa.,
.lamda.), an IgG2b antibody (e.g. IgG2b, .kappa., .lamda.), an IgG3
antibody (e.g. IgG3, .kappa., .lamda.) or an IgG4 antibody (e.g.
IgG4, .kappa., .lamda.).
[0187] As used herein, a "heterologous antibody" is defined in
relation to a transgenic organism producing such an antibody. This
term refers to an antibody having an amino acid sequence or an
encoding nucleic acid sequence corresponding to that found in an
organism not consisting of the transgenic organism, and being
generally derived from a species other than the transgenic
organism.
[0188] As used herein, a "heterohybrid antibody" refers to an
antibody having light and heavy chains of different organismal
origins. For example, an antibody having a human heavy chain
associated with a murine light chain is a heterohybrid
antibody.
[0189] The antibodies described herein are preferably isolated. An
"isolated antibody" as used herein, 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 CLDN18.2 is substantially free of antibodies
that specifically bind antigens other than CLDN18.2). An isolated
antibody that specifically binds to an epitope, isoform or variant
of human CLDN18.2 may, however, have cross-reactivity to other
related antigens, e.g., from other species (e.g., CLDN18.2 species
homologs). Moreover, an isolated antibody may be substantially free
of other cellular material and/or chemicals. In one embodiment of
the invention, a combination of "isolated" monoclonal antibodies
relates to antibodies having different specificities and being
combined in a well defined composition or mixture.
[0190] 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 antibody can be performed by
fragments of a full-length antibody. 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
fragments 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 linker. Furthermore, although the two domains of the Fv
fragment, 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 al. (1988) Science 242: 423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding fragment" of an antibody. A
further example is binding-domain immunoglobulin fusion proteins
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 variable region or a light chain variable region. The
binding-domain immunoglobulin fusion proteins are further disclosed
in US 2003/0118592 and US 2003/0133939. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies.
[0191] 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 structure/antigen/epitope. Thus,
the binding domain according to the invention designates an
"antigen-interaction-site".
[0192] All antibodies and derivatives of antibodies such as
antibody fragments as described herein for the purposes of the
invention are encompassed by the term "antibody". The term
"antibody derivatives" refers to any modified form of an antibody,
e.g., a conjugate of the antibody and another agent or antibody, or
an antibody fragment. Furthermore, the antibodies and derivatives
of antibodies as described herein are useful for producing binding
agents of the invention such as antibody fragments.
[0193] Naturally occurring antibodies are generally monospecific,
i.e. they bind to a single antigen. The present invention also
envisions binding agents which are bispecific or multispecific
molecules binding to CLDN18.2 and to one or more further antigens.
Particularly preferred are binding agents binding to a cytotoxic
cell (e.g. by engaging the CD3 receptor) and a cancer cell (by
engaging CLDN18.2). Such binding agents are at least bispecific or
multispecific such as trispecific, tetraspecific and so on.
[0194] Thus, in one embodiment, a binding agent according to the
invention comprises at least two binding domains, wherein a first
binding domain binds to CLDN18.2 and a second binding domain binds
to CD3. Such binding agent may bind to a cytotoxic cell (e.g. by
engaging the CD3 receptor) and a cancer cell expressing CLDN18.2 to
be destroyed as a target.
[0195] The bispecific or multispecific binding agent may be in the
format of an antibody molecule or of an antibody-like molecule or
of a protein scaffold with antibody-like properties or of a cyclic
peptide with at least two binding specificities. Thus, the binding
agent may comprise one or more antibodies as described herein or
fragments thereof.
[0196] According to the invention, a bispecific molecule, in
particular a bispecific protein, such as a bispecific antibody is a
molecule that has two different binding specificities and thus may
bind to two different types of antigen such as CLDN18.2 and CD3.
Particularly, the term "bispecific antibody" as used herein refers
to an antibody comprising two antigen-binding sites, a first
binding site having affinity for a first antigen or epitope and a
second binding site having binding affinity for a second antigen or
epitope distinct from the first. In particular, a bispecific
antibody is an artificial protein that is composed of fragments of
two different antibodies (said fragments of two different
antibodies forming two binding domains) and consequently binds to
two different types of antigen. A bispecific antibody preferably is
engineered to simultaneously bind to an immune cell, such as an
immune effector cell, in particular a T cell such as a cytotoxic
cell (e.g. by binding to CD3) and a target cell like a cancer cell
(by binding to the tumor-associated antigen CLDN18.2) to be
destroyed.
[0197] The term "bispecific antibody" also includes diabodies.
Diabodies are bivalent, bispecific antibodies in which VH and VL
domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994)
Structure 2: 1121-1123).
[0198] "Multispecific binding agents" are molecules which have more
than two different binding specificities.
[0199] Particularly preferred according to the invention are
bispecific antibodies including bispecific antibody fragments, in
particular bispecific single chain antibodies including bispecific
single chain antibody fragments. The term "bispecific single chain
antibody" denotes a single polypeptide chain comprising two binding
domains. In particular, the term "bispecific single chain antibody"
or "single chain bispecific antibody" or related terms in
accordance with the present invention preferably mean antibody
constructs resulting from joining at least two antibody variable
regions in a single polypeptide chain devoid of the constant and/or
Fc portion(s) present in full immunoglobulins.
[0200] For example, a bispecific single chain antibody may be a
construct with a total of two antibody variable regions, for
example two VH regions, each capable of specifically binding to a
separate antigen, and connected with one another through a short
polypeptide spacer such that the two antibody variable regions with
their interposed spacer exist as a single contiguous polypeptide
chain. Another example of a bispecific single chain antibody may be
a single polypeptide chain with three antibody variable regions.
Here, two antibody variable regions, for example one VH and one VL,
may make up an scFv, wherein the two antibody variable regions are
connected to one another via a synthetic polypeptide linker, the
latter often being genetically engineered so as to be minimally
immunogenic while remaining maximally resistant to proteolysis.
This scFv is capable of specifically binding to a particular
antigen, and is connected to a further antibody variable region,
for example a VH region, capable of binding to a different antigen
than that bound by the scFv. Yet another example of a bispecific
single chain antibody may be a single polypeptide chain with four
antibody variable regions. Here, the first two antibody variable
regions, for example a VH region and a VL region, may form one scFv
capable of binding to one antigen, whereas the second VH region and
VL region may form a second scFv capable of binding to another
antigen. Within a single contiguous polypeptide chain, individual
antibody variable regions of one specificity may advantageously be
separated by a synthetic polypeptide linker, whereas the respective
scFvs may advantageously be separated by a short polypeptide spacer
as described above.
[0201] According to one embodiment of the invention, the first
binding domain of the bispecific antibody comprises one antibody
variable domain, preferably a VHH domain. According to one
embodiment of the invention, the first binding domain of the
bispecific antibody comprises two antibody variable domains,
preferably a scFv, i.e. VH-VL or VL-VH. According to one embodiment
of the invention, the second binding domain of the bispecific
antibody comprises one antibody variable domain, preferably a VHH
domain. According to one embodiment of the invention, the second
binding domain of the bispecific antibody comprises two antibody
variable domains, preferably a scFv, i.e. VH-VL or VL-VH. In its
minimal form, the total number of antibody variable regions in the
bispecific antibody according to the invention is thus only two.
For example, such an antibody could comprise two VH or two VHH
domains.
[0202] According to one embodiment of the invention, the first
binding domain and the second binding domain of the bispecific
antibody each comprise one antibody variable domain, preferably a
VHH domain. According to one embodiment of the invention, the first
binding domain and the second binding domain of the bispecific
antibody each comprise two antibody variable domains, preferably a
scFv, i.e. VH-VL or VL-VH. In this embodiment, the binding agent of
the invention preferably comprises (i) a heavy chain variable
domain (VH) of a CLDN18.2 antibody, (ii) a light chain variable
domain (VL) of a CLDN18.2 antibody, (iii) a heavy chain variable
domain (VH) of an antibody to a second antigen, e.g. of a CD3
antibody and (iv) a light chain variable domain (VL) of an antibody
to a second antigen, e.g. of a CD3 antibody.
[0203] Bispecific full-length antibodies may be obtained by
covalently linking two monoclonal antibodies or by conventional
hybrid-hybridoma techniques. Covalent linking of two monoclonal
antibodies is described in Anderson, Blood 80 (1992), 2826-34. In
the context of this invention, one of the antibodies may be
specific for CLDN18.2 and the other one for CD3.
[0204] In one embodiment, the bispecific binding agent is in the
format of an antibody-like molecule with a heavy chain containing
two consecutive N-terminal variable domains with different
specificities and a light chain with two consecutive variable
domains with different specificities resulting in four binding
domains with two different specificities (Wu et al., Nat.
Biotechnology, 2007, 25(11)), wherein one specificity may be CD3
and the other specificity is CLDN18.2.
[0205] In a preferred embodiment, the bispecific binding agent of
the invention is in the format of an antibody fragment.
[0206] In one embodiment, the bispecific molecules according to the
invention comprises two Fab regions, e.g. one being directed
against CLDN18.2 and the other being directed against CD3. In one
embodiment, the molecule of the invention is an antigen binding
fragment (Fab)2 complex. The Fab2 complex is composed of two Fab
fragments, one Fab fragment comprising a Fv domain, i.e. VH and VL
domains, e.g. specific for a CD3 antigen, and the other Fab
fragment comprising a Fv domain specific for CLDN18.2. Each of the
Fab fragments may be composed of two single chains, a VL-CL module
and a VH-CH module. Alternatively, each of the individual Fab
fragments may be arranged in a single chain, preferably,
VL-CL-CH-VH, and the individual variable and constant domains may
be connected with a peptide linker. In general, the individual
single chains and Fab fragments may be connected via disulfide
bonds, adhesive domains, chemically linked and/or peptide linker.
The bispecific molecule may also comprise more than two Fab
fragments, in particular, the molecule may be a Fab3, Fab4, or a
multimeric Fab complex with specificity for 2, 3, 4, or more
different antigens. The invention also includes chemically linked
Fabs.
[0207] In one embodiment, the binding agent according to the
invention includes various types of bivalent and trivalent
single-chain variable fragments (scFvs), fusion proteins mimicking
the variable domains of two antibodies. A single-chain variable
fragment (scFv) is a fusion protein of the variable regions of the
heavy (VH) and light chains (VL) of immunoglobulins, connected with
a short linker peptide of ten to about 25 amino acids. The linker
is usually rich in glycine for flexibility, as well as serine or
threonine for solubility, and can either connect the N-terminus of
the VH with the C-terminus of the VL, or vice versa. Divalent (or
bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can
be engineered by linking two scFvs. This can be done by producing a
single peptide chain with two VH and two VL regions, yielding
tandem scFvs. The invention also includes multispecific molecules
comprising more than two scFvs binding domains. This makes it
possible that the molecule comprises either multiple antigen
specificities and is a trispecific, tetraspecific, or multispecific
molecule, or the molecule is a bispecific molecule comprising more
than one scFv binding domain with specificity for the same antigen.
In particular, the molecule of the invention may be a multispecific
single chain Fv.
[0208] Another possibility is the creation of scFvs with linker
peptides that are too short for the two variable regions to fold
together (about five amino acids), forcing scFvs to dimerize. This
type is known as diabodies. Still shorter linkers (one or two amino
acids) lead to the formation of trimers, so-called triabodies or
tribodies. Tetrabodies have also been produced. They exhibit an
even higher affinity to their targets than diabodies.
[0209] A particularly preferred example of a bispecific antibody
fragment is a diabody (Kipriyanov, Int. J. Cancer 77 (1998),
763-772), which is a small bivalent and bispecific antibody
fragment. Diabodies comprise a heavy chain variable domain (VH)
connected to a light chain variable domain (VL) on the same
polypeptide chain (VH-VL) connected by a peptide linker that is too
short to allow pairing between the two domains on the same chain.
This forces pairing with the complementary domains of another chain
and promotes the assembly of a dimeric molecule with two functional
antigen binding sites. To construct bispecific diabodies, the
V-domains of e.g. an anti-CD3 antibody and an anti-CLDN18.2
antibody may be fused to create the two chains
VH(CD3)-VL(CLDN18.2), VH(CLDN18.2)-VL(CD3). Each chain by itself is
not able to bind to the respective antigen, but recreates the
functional antigen binding sites on pairing with the other chain.
To this end, a peptide linker that is too short to allow pairing
between the two domains on the same chain is used. The two scFv
molecules, with a linker between heavy chain variable domain and
light chain variable domain that is too short for intramolecular
dimerization, are co-expressed and self assemble to form
bi-specific molecules with the two binding sites at opposite
ends.
[0210] In one embodiment, the multispecific molecule according to
the invention comprises variable (VH, VL) and constant domains (C)
of immunoglobulins. In one embodiment the bispecific molecule is a
minibody, preferably, a minibody comprising two single VH-VL-C
chains that are connected with each other via the constant domains
(C) of each chain. According to this aspect, the corresponding
variable heavy chain regions (VH), corresponding variable light
chain regions (VL) and constant domains (C) may be arranged, from
N-terminus to C-terminus, in the order
VH(CLDN18.2)-VL(CLDN18.2)-(C) and VH(CD3)-VL(CD3)-C, wherein C is
preferably a CH3 domain. Pairing of the constant domains results in
formation of the minibody.
[0211] According to another particularly preferred aspect, the
bispecific binding agent of the invention is in the format of a
bispecific single chain antibody construct, whereby said construct
comprises or consists of at least two binding domains, whereby one
of said domains binds to CLDN18.2 and a second domain binds to
another antigen, e.g. CD3. Such molecules, also termed "bispecific
T cell engagers" (BiTEs; the term BiTE only refers to bi-specific
molecules of which one arm is specific for CD3) consist of two scFv
molecules connected via a linker peptide.
[0212] As used herein, a "bispecific single chain antibody" denotes
a single polypeptide chain comprising two binding domains. Each
binding domain comprises one variable region from an antibody heavy
chain ("VH region"), wherein the VH region of the first binding
domain specifically binds to the CLDN18.2, and the VH region of the
second binding domain specifically binds to another antigen, e.g.
CD3. The two binding domains are optionally linked to one another
by a short polypeptide spacer. A non-limiting example for a
polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) (SEQ ID NO:
7) and repeats thereof. Each binding domain may additionally
comprise one variable region from an antibody light chain ("VL
region"), the VH region and VL region within each of the first and
second binding domains being linked to one another via a
polypeptide linker long enough to allow the VH region and VL region
of the first binding domain and the VH region and VL region of the
second binding domain to pair with one another.
[0213] According to this aspect, the corresponding variable heavy
chain regions (VH) and the corresponding variable light chain
regions (VL) are arranged, from N-terminus to C-terminus, in the
order VH(CLDN18.2)-VL(CLDN18.2)-VH(CD3)-VL(CD3),
VH(CD3)-VL(CD3)-VH(CLDN18.2)-VL(CLDN18.2) or
VH(CD3)-VL(CD3)-VL(CLDN18.2)-VH(CLDN18.2). It is, however, also
envisaged that the bispecific single chain antibodies of the
invention comprise other domain arrangements, such as
VL(CLDN18.2)-VH(CLDN18.2)-VH(CD3)-VL(CD3),
VL(CLDN18.2)-VH(CLDN18.2)-VL(CD3)-VH(CD3),
VH(CLDN18.2)-VL(CLDN18.2)-VL(CD3)-VH(CD3),
VL(CD3)-VH(CD3)-VH(CLDN18.2)-VL(CLDN18.2),
VL(CD3)-VH(CD3)-VL(CLDN18.2)-VH(CLDN18.2). "CD3" is only given
herein as an example to designate a second binding specificity to
an antigen.
[0214] A long linker generally connects the corresponding variable
heavy chain regions (VH) and the corresponding variable light chain
regions (VL) to create a scFv binding domain while a short linker
generally connects two scFv binding domains. The linker is
generally designed to provide flexibility and protease resistance,
and preferably, the linker comprises glycine and/or serine amino
acid residues. Short peptide linkers may consist of 12 or less such
as 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably, 5
or 6 amino acids. Short peptide linkers preferably comprise the
amino acid sequences SGGGGS (SEQ ID NO: 6) or GGGGS (SEQ ID NO: 7).
Long peptide linkers may consist of 12 or more, such as 15 to 25 or
15 to 20 or 15 to 18 amino acids. Long peptide linkers preferably
comprise the amino acid sequences (GGGGS)3 (SEQ ID NO: 4) or
VE(GGSGGS)2GGVD (SEQ ID NO: 8). Further long peptide linkers may
comprise the amino acid sequences (GGGGS)4 (SEQ ID NO: 9), (GGGGS)5
(SEQ ID NO: 10) or GGGGS(GGS)3GGGS (SEQ ID NO: 11).
[0215] Binding agents according to the invention may also comprises
an amino acid sequence for facilitating secretion of the molecule,
such as a N-terminal secretion signal, and/or one or more epitope
tags facilitating binding, purification or detection of the
molecule.
[0216] Preferably, the secretion signal is a signal sequence that
allows a sufficient passage through the secretory pathway and/or
secretion of the binding agent into the extracellular environment.
Preferably, the secretion signal sequence is cleavable and is
removed from the mature binding agent. The secretion signal
sequence preferably is chosen with respect to the cell or organism
wherein the binding agent is produced in.
[0217] The amino acid sequence of an epitope tag may be introduced
to any position within the amino acid sequence of the binding
agent, and may take the shape of a loop within the encoded protein
structure, or it may be N-terminally or C-terminally fused to the
binding agent. Preferably, the epitope tag is C-terminally fused to
the binding agent. The epitope tag may contain a cleavage site that
allows a removal of the tag from the binding agent. Said epitope
tag can be any kind of epitope tag that is functional under native
and/or denaturing conditions, preferable a histidin tag, most
preferable a tag comprising six histidins.
[0218] The bispecific binding agent of the invention may contain,
in addition to said first and second binding domain, a further
binding domain which serves e.g. to enhance selectivity for tumor
cells. This can be achieved e.g. by providing binding domains that
bind to other antigens expressed on tumor cells.
[0219] According to the invention, the term "binding agent to
CLDN18.2" further includes chimeric antigen receptors (CAR).
According to the invention the term "chimeric antigen receptor
(CAR)" is synonymous with the terms "chimeric T cell receptor" and
"artificial T cell receptor".
[0220] 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 receptor may 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 artificial 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.
[0221] 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.
[0222] Following antigen recognition, receptors cluster and a
signal is transmitted to the cell. The most commonly used
endodomain component is CD3-zeta. This transmits an activation
signal to the T cell after antigen is bound.
[0223] Adoptive cell transfer therapy with CAR-engineered T cells
expressing chimeric antigen receptors is a promising anti-cancer
therapeutic as CAR-modified T cells can be engineered to target
virtually any tumor antigen. For example, patient's T cells may be
genetically engineered to express CARs specifically directed
towards antigens on the patient's tumor cells, then infused back
into the patient.
[0224] In the context of the present invention, the binding agents
are preferably therapeutically effective and/or are capable of
eliciting immune effector functions as described herein.
Preferably, said immune effector functions are directed against
cells carrying the tumor antigen CLDN18.2 on their surface such as
cancer cells.
[0225] The term "therapeutically effective" relates to a
therapeutic effectiveness when administered to an individual. The
term "therapeutically effective" further relates to the ability to
change, preferably cure, alleviate or partially arrest the clinical
manifestations of a given disease and its complications in a
therapeutic intervention.
[0226] The term "immune effector functions" in the context of the
present invention includes any functions mediated by components of
the immune system that result e.g. in the inhibition of tumor
growth and/or inhibition of tumor development, including inhibition
of tumor dissemination and metastasis. Preferably, immune effector
functions result in killing of tumor cells. Such functions comprise
complement dependent cytotoxicity (CDC), antibody-dependent
cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated
phagocytosis (ADCP), induction of apoptosis in the cells carrying
the tumor-associated antigen, cytolysis of the cells carrying the
tumor-associated antigen, and/or inhibition of proliferation of the
cells carrying the tumor-associated antigen. Binding agents may
also exert an effect simply by binding to tumor-associated antigens
on the surface of a cancer cell. For example, antibodies may block
the function of the tumor-associated antigen or induce apoptosis
just by binding to the tumor-associated antigen on the surface of a
cancer cell.
[0227] ADCC describes the cell-killing ability of effector cells,
in particular lymphocytes, which preferably requires the target
cell being marked by an antibody.
[0228] ADCC preferably occurs when antibodies bind to antigens on
tumor cells and the antibody Fc domains engage Fc receptors (FcR)
on the surface of immune effector cells. Several families of Fc
receptors have been identified, and specific cell populations
characteristically express defined Fc receptors. ADCC can be viewed
as a mechanism to directly induce a variable degree of immediate
tumor destruction that leads to antigen presentation and the
induction of tumor-directed T-cell responses. Preferably, in vivo
induction of ADCC will lead to tumor-directed T-cell responses and
host-derived antibody responses.
[0229] CDC is another cell-killing method that can be directed by
antibodies. IgM is the most effective isotype for complement
activation. IgG1 and IgG3 are also both very effective at directing
CDC via the classical complement-activation pathway. Preferably, in
this cascade, the formation of antigen-antibody complexes results
in the uncloaking of multiple C1q binding sites in close proximity
on the CH2 domains of participating antibody molecules such as IgG
molecules (C1q is one of three subcomponents of complement C1).
Preferably these uncloaked C1q binding sites convert the previously
low-affinity C1q-IgG interaction to one of high avidity, which
triggers a cascade of events involving a series of other complement
proteins and leads to the proteolytic release of the effector-cell
chemotactic/activating agents C3a and C5a. Preferably, the
complement cascade ends in the formation of a membrane attack
complex, which creates pores in the cell membrane that facilitate
free passage of water and solutes into and out of the cell.
[0230] The binding agents described herein may be conjugated to a
therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radioisotope. A cytotoxin or cytotoxic
agent includes any agent that is detrimental to and, in particular,
kills cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Suitable therapeutic agents for forming conjugates
include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
fludarabin, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU)
and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II)
(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC), and anti-mitotic agents (e.g., vincristine and vinblastine).
In a preferred embodiment, the therapeutic agent is a cytotoxic
agent or a radiotoxic agent. In another embodiment, the therapeutic
agent is an immunosuppressant. In yet another embodiment, the
therapeutic agent is GM-CSF. In a preferred embodiment, the
therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate,
carmustine, chlorambucil, cyclophosphamide or ricin A.
[0231] Binding agents also can be conjugated to a radioisotope,
e.g., iodine-131, yttrium-90 or indium-111, to generate cytotoxic
radiopharmaceuticals.
[0232] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62: 119-58 (1982).
[0233] The term "binding" according to the invention preferably
relates to a specific binding.
[0234] According to the present invention, an agent such as 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 (K.sub.D). Preferably, the term "significant affinity"
refers to the binding to a predetermined target with a dissociation
constant (K.sub.D) of 10.sup.-5 M or lower, 10.sup.-6 M or lower,
10.sup.-7 M or lower, 10.sup.-8M or lower, 10.sup.-9M or lower,
10.sup.-19M or lower, 10.sup.-11M or lower, or 10.sup.-12M or
lower.
[0235] 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
.mu.g/ml or higher. Preferably, an agent has no significant
affinity for a target if it binds to said target with a K.sub.D
that is at least 10-fold, 100-fold, 10.sup.3-fold, 10.sup.4-fold,
10.sup.5-fold, or 10.sup.6-fold higher than the K.sub.D for binding
to the predetermined target to which the agent is capable of
binding. For example, if the K.sub.D for binding of an agent to the
target to which the agent is capable of binding is 10.sup.-7 M, the
K.sub.D for binding to a target for which the agent has no
significant affinity would be at least 10.sup.-6 M, 10.sup.-5 M,
10.sup.-4 M, 10.sup.-3 M, 10.sup.-2 M, or 10.sup.-1M.
[0236] An agent such as an antibody is specific for a predetermined
target if it is capable of binding to said predetermined target
while it is not capable of binding to other targets, i.e. has no
significant affinity for other targets and does not significantly
bind to other targets in standard assays. According to the
invention, an agent is specific for CLDN18.2 if it is capable of
binding to CLDN18.2 but is not (substantially) capable of binding
to other targets. Preferably, an agent is specific for CLDN18.2 if
the affinity for and the binding to such other targets does not
significantly exceed the affinity for or binding to
CLDN18.2-unrelated proteins such as bovine serum albumin (BSA),
casein, human serum albumin (HSA) or non-claudin transmembrane
proteins such as MHC molecules or transferrin receptor or any other
specified polypeptide. Preferably, an agent is specific for a
predetermined target if it binds to said target with a K.sub.D that
is at least 10-fold, 100-fold, 10.sup.3-fold, 10.sup.4-fold,
10.sup.5-fold, or 10.sup.6-fold lower than the K.sub.D for binding
to a target for which it is not specific. For example, if the
K.sub.D for binding of an agent to the target for which it is
specific is 10.sup.-7 M, the K.sub.D for binding to a target for
which it is not specific would be at least 10.sup.-6 M, 10.sup.-5
M, 10.sup.4 M, 10.sup.-3 M, 10.sup.-2 M, or 10.sup.4 M.
[0237] Binding of an agent to a target can be determined
experimentally using any suitable method; see, for example,
Berzofsky et al., "Antibody-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 methods described herein. Affinities may be readily
determined using conventional techniques, such as by equilibrium
dialysis; by using surface plasmon resonance analytic (e.g.
Biacore), 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 different
conditions, e.g., salt concentration, pH. Thus, measurements of
affinity and other antigen-binding parameters, e.g., K.sub.D,
IC.sub.50, are preferably made with standardized solutions of
antibody and antigen, and a standardized buffer.
[0238] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes.
[0239] As used herein, "isotype switching" refers to the phenomenon
by which the class, or isotype, of an antibody changes from one Ig
class to one of the other Ig classes.
[0240] The term "naturally occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally occurring.
[0241] The term "rearranged" as used herein refers to a
configuration of a heavy chain or light chain immunoglobulin locus
wherein a V segment is positioned immediately adjacent to a D-J or
J segment in a conformation encoding essentially a complete VH or
VL domain, respectively. A rearranged immunoglobulin (antibody)
gene locus can be identified by comparison to germline DNA; a
rearranged locus will have at least one recombined heptamer/nonamer
homology element.
[0242] The term "unrearranged" or "germline configuration" as used
herein in reference to a V segment refers to the configuration
wherein the V segment is not recombined so as to be immediately
adjacent to a D or J segment.
[0243] In one embodiment, a binding agent of the invention has the
ability of binding to CLDN18.2, i.e. the ability of binding to an
epitope present in CLDN18.2, preferably an epitope located within
the extracellular domains of CLDN18.2, in particular the first
extracellular loop, preferably amino acid positions 29 to 78 of
CLDN18.2. In particular embodiments, an agent having the ability of
binding to CLDN18.2 binds to an epitope on CLDN18.2 which is not
present on CLDN18.1.
[0244] An agent having the ability of binding to CLDN18.2
preferably binds to CLDN18.2 but not to CLDN18.1. Preferably, an
agent having the ability of binding to CLDN18.2 is specific for
CLDN18.2. Preferably, an agent having the ability of binding to
CLDN18.2 binds to CLDN18.2 expressed on the cell surface. In
particular preferred embodiments, an agent having the ability of
binding to CLDN18.2 binds to native epitopes of CLDN18.2 present on
the surface of living cells.
[0245] In a preferred embodiment, a binding domain for CLDN18.2 or
binding agent to CLDN18.2 comprises the following combination of
heavy chain variable region (VH) and light chain variable region
(VL): The VH comprises an amino acid sequence represented by SEQ ID
NO: 2 or a fragment thereof and the VL comprises an amino acid
sequence represented by SEQ ID NO: 3 or a fragment thereof.
[0246] In a preferred embodiment, a binding domain for CLDN18.2 or
binding agent to CLDN18.2 comprises the following combination of
heavy chains and light chains: The heavy chain comprises an amino
acid sequence represented by SEQ ID NO: 12 or a fragment thereof
and the light chain comprises an amino acid sequence represented by
SEQ ID NO: 13 or a fragment thereof.
[0247] In a preferred embodiment, a binding domain for CLDN18.2 or
binding agent to CLDN18.2 comprises the following set of
complementarity-determining regions CDR1, CDR2 and CDR3: VH: CDR1:
positions 45-52 of SEQ ID NO: 12, CDR2: positions 70-77 of SEQ ID
NO: 12, CDR3: positions 116-126 of SEQ ID NO: 12, VL: CDR1:
positions 47-58 of SEQ ID NO: 13, CDR2: positions 76-78 of SEQ ID
NO: 13, CDR3: positions 115-123 of SEQ ID NO: 13.
[0248] 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 regions (CDRs)
are selected from a set of complementarity-determining regions
CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the
term "fragment" refers to the complementarity-determining regions
CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or
of the light chain variable region (VL).
[0249] In one embodiment a binding agent 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 intervening
framework regions. Preferably, the portion will also include at
least about 50% of either or both of the first and fourth framework
regions, the 50% being the C-terminal 50% of the first framework
region and the N-terminal 50% of the fourth framework region.
Construction of binding agents made by recombinant DNA techniques
may result in the introduction of residues N- or C-terminal to the
variable regions encoded by linkers introduced to facilitate
cloning or other manipulation steps, including the introduction of
linkers to join variable regions of the invention to further
protein sequences including immunoglobulin heavy chains, other
variable domains (for example in the production of diabodies) or
protein labels.
[0250] In one embodiment a binding agent 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.
[0251] The CD3 (cluster of differentiation 3) complex denotes an
antigen that is expressed on mature human T-cells, thymocytes and a
subset of natural killer cells as part of the multimolecular T-cell
receptor (TCR) complex. The T-cell co-receptor is a protein complex
and is composed of four distinct chains. In mammals, the complex
contains a CD3.gamma. chain, a CD3.delta. chain, and two
CD3.epsilon. chains. These chains associate with a molecule known
as the T-cell receptor (TCR) and the .zeta.-chain to generate an
activation signal in T lymphocytes. The TCR, .zeta.-chain, and CD3
molecules together comprise the TCR complex.
[0252] CD3 is responsible for the signal transduction of the TCR.
As described by Lin and Weiss, Journal of Cell Science 114, 243-244
(2001), activation of the TCR complex by binding of MHC-presented
specific antigen epitopes results in the phosphorylation of
immunoreceptor tyrosine-based activation motifs (ITAMs) by Src
family kinases, triggering recruitment of further kinases which
results in T cell activation including Ca.sup.2+ release.
Clustering of CD3 on T cells, e.g. by immobilized
anti-CD3-antibodies, leads to T cell activation similar to the
engagement of the T cell receptor, but independent from its clone
typical specificity.
[0253] As used herein, "CD3" includes human CD3 and denotes an
antigen that is expressed on human T cells as part of the
multimolecular T cell receptor complex.
[0254] With respect to CD3, the binding agent of the invention
preferably recognizes the epsilon-chain of CD3, particular, it
recognizes an epitope that corresponds to the first 27 N-terminal
amino acids of CD3 epsilon or functional fragments of this 27 amino
acid stretch.
[0255] Anti-CD3 antibodies which are useful for providing binding
agents binding to CD3 include but are not limited to UCHT1-HS
(humanized mAB), UCHT1-MM (murine mAB), CLB-T3, TR66, 145-2C11.
[0256] UCHT1 is a monoclonal IgG1 anti-CD3 monoclonal antibody
which detects CD3 in human and primate sample types. CLB-T3 is a
mouse monoclonal anti-CD3 antibody which is directed against the
CD3 antigen and reacts with 80-90% human peripheral T lymphocytes
and medullary thymocytes. TR66 is a mouse IgG1 monoclonal anti-CD3
antibody which recognizes the epsilon-chain of human CD3. 145-2C11
is an armenian hamster monoclonal anti-mouse CD3 antibody.
[0257] Preferably, the VH and VL regions of the CD3-binding domain
are derived from antibodies/antibody molecules and antibody-like
molecules which are capable of specifically recognizing the human
CD3 in the context of other TCR subunits as present on activated
primary human T cells expressing the TCR in its native
configuration. The VH and VL regions derived from an antibody
specific for the CD3-epsilon chain are most preferred and said
(parental) antibodies should be capable of specifically binding
epitopes reflecting the native or near-native structure or a
conformational epitope of human CD3 presented in the context of the
TCR complex. In a preferred embodiment of the invention, the VH and
VL regions of the CD3-binding domain are derived from a CD3
specific antibody selected from the group consisting of UCHT1-HS,
UCHT1-MM, CLB-T3 and TR66, preferably TR66.
[0258] Antibodies described herein for e.g. providing VL and VH
regions can be produced by a variety of techniques, including
conventional monoclonal antibody methodology, e.g., the standard
somatic cell hybridization technique of Kohler and Milstein, Nature
256: 495 (1975). Although somatic cell hybridization procedures are
preferred, in principle, other techniques for producing monoclonal
antibodies can be employed, e.g., viral or oncogenic transformation
of B-lymphocytes or phage display techniques using libraries of
antibody genes.
[0259] The preferred animal system for preparing hybridomas that
secrete monoclonal antibodies is the murine system. Hybridoma
production in the mouse is a very well established procedure.
Immunization protocols and techniques for isolation of immunized
splenocytes for fusion are known in the art. Fusion partners (e.g.,
murine myeloma cells) and fusion procedures are also known.
[0260] Other preferred animal systems for preparing hybridomas that
secrete monoclonal antibodies are the rat and the rabbit system
(e.g. described in Spieker-Polet et al., Proc. Natl. Acad. Sci.
U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol.
124: 295 (2005)).
[0261] To generate antibodies, 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 preparation of the antigen do not
result in antibodies, mice can also be immunized with cells
expressing the antigen, e.g., a cell line, to promote immune
responses.
[0262] The immune response 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 intravenously with antigen expressing cells 3
days before sacrifice and removal of the spleen to increase the
rate of specific antibody secreting hybridomas.
[0263] To generate hybridomas producing monoclonal antibodies,
splenocytes and lymph node cells from immunized mice can be
isolated and fused to an appropriate immortalized cell line, such
as a mouse myeloma cell line. The resulting hybridomas can then be
screened for the production of antigen-specific antibodies.
Individual wells can then be screened by ELISA for antibody
secreting hybridomas. By Immunofluorescence and FACS analysis using
antigen expressing cells, antibodies with specificity for the
antigen can be identified. The antibody secreting hybridomas can be
replated, screened again, and if still positive for monoclonal
antibodies can be subcloned by limiting dilution. The stable
subclones can then be cultured in vitro to generate antibody in
tissue culture medium for characterization.
[0264] Nonlabeled murine antibodies are highly immunogenic in man
when repetitively applied leading to reduction of the therapeutic
effect. The main immunogenicity is mediated by the heavy chain
constant regions. The immunogenicity of murine antibodies in man
can be reduced or completely avoided if respective antibodies are
chimerized or humanized. Chimeric antibodies are antibodies, the
different portions of which are derived from different animal
species, such as those having a variable region derived from a
murine antibody and a human immunoglobulin constant region.
Chimerisation of antibodies is achieved by joining of the variable
regions of the murine antibody heavy and light chain with the
constant region of human heavy and light chain (e.g. as described
by Kraus et al., in Methods in Molecular Biology series,
Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a
preferred embodiment chimeric antibodies are generated by joining
human kappa-light chain constant region to murine light chain
variable region. In an also preferred embodiment chimeric
antibodies can be generated by joining human lambda-light chain
constant region to murine light chain variable region. The
preferred heavy chain constant regions for generation of chimeric
antibodies are IgG1, IgG3 and IgG4. Other preferred heavy chain
constant regions for generation of chimeric antibodies are IgG2,
IgA, IgD and IgM.
[0265] Antibodies interact with target antigens predominantly
through amino acid residues that are located in the six heavy and
light chain complementarity determining regions (CDRs). For this
reason, the amino acid sequences within CDRs are more diverse
between individual antibodies than sequences outside of CDRs.
Because CDR sequences are responsible for most antibody-antigen
interactions, it is possible to express recombinant antibodies that
mimic the properties of specific naturally occurring antibodies by
constructing expression vectors that include CDR sequences from the
specific naturally occurring antibody grafted onto framework
sequences from a different antibody with different properties (see,
e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et
al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc.
Natl. Acad. Sci. U.S.A 86: 10029-10033). Such framework sequences
can be obtained from public DNA databases that include germline
antibody gene sequences. These germline sequences will differ from
mature antibody gene sequences because they will not include
completely assembled variable genes, which are formed by V (D) J
joining during B cell maturation. Germline gene sequences will also
differ from the sequences of a high affinity secondary repertoire
antibody at individual evenly across the variable region.
[0266] It is to be understood that the peptide mimotopes and/or
binding agents described herein may be delivered to a patient by
administering a nucleic acid such as RNA encoding the peptide
mimotope and/or binding agent and/or by administering a host cell
comprising a nucleic acid such as RNA encoding the peptide mimotope
and/or binding agent. Thus, a nucleic acid encoding a peptide
mimotope and/or binding agent when administered to a patient may be
present in naked form or in a suitable delivery vehicle such as in
the form of liposomes or viral particles, or within a host cell.
The nucleic acid provided can produce the peptide mimotope and/or
binding agent over extended time periods in a sustained manner.
Nucleic acids to be delivered to a patient can be produced by
recombinant means. If a nucleic acid is administered to a patient
without being present within a host cell, it is preferably taken up
by cells of the patient for expression of the peptide mimotope
and/or binding agent encoded by the nucleic acid. If a nucleic 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 peptide mimotope and/or binding agent
encoded by the nucleic acid.
[0267] The term "recombinant" in the context of the present
invention means "made through genetic engineering". Preferably, a
"recombinant object" such as a recombinant nucleic acid in the
context of the present invention is not occurring naturally.
[0268] 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.
[0269] The term "nucleic acid", as used herein, 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 synthetic RNA.
[0270] 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 adenoviral or
baculoviral vectors, or artificial chromosome vectors such as
bacterial artificial chromosomes (BAC), yeast artificial
chromosomes (YAC), or P1 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 sequence 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 amplify a certain desired DNA
fragment and may lack functional sequences needed for expression of
the desired DNA fragments.
[0271] 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
ribonucleotide residues. "Ribonucleotide" relates to a nucleotide
with a hydroxyl group at the 2'-position of a (3-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 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 include addition 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 deoxynucleotides. These
altered RNAs can be referred to as analogs or analogs of
naturally-occurring RNA.
[0272] 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 produced using DNA as
template and encodes a peptide or protein. mRNA typically comprises
a 5' non translated region (5'-UTR), a protein or peptide coding
region and a 3' non translated region (3'-UTR). 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 methodology is known
to the skilled person. For example, there is a variety of in vitro
transcription kits commercially available.
[0273] 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 embodiment, 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 self-replicating RNA
is a viral gene expression vector. In one embodiment, the virus is
Semliki forest virus. In one embodiment, the self-replicating RNA
contains one or more transgenes at least one of said transgenes
encoding the peptide mimotope/binding agent 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
embodiment, the self-replicating RNA is in vitro transcribed
RNA.
[0274] 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.
[0275] 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.
[0276] In one embodiment of the invention, the RNA used according
to the invention does not have uncapped 5'-triphosphates. Removal
of such uncapped 5'-triphosphates can be achieved by treating RNA
with a phosphatase.
[0277] 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
pseudouridine is substituted partially or completely, preferably
completely, for uridine.
[0278] In one embodiment, the term "modification" 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 5'-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
5'-cap analog that resembles the RNA cap structure and is modified
to possess the ability to stabilize RNA if attached thereto,
preferably in vivo and/or in a cell.
[0279] Providing an RNA with a 5'-cap or 5'-cap analog may be
achieved by in vitro transcription of a DNA template 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 vaccinia
virus.
[0280] The RNA may comprise further modifications. For example, a
further 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 introduction of a UTR which is not related 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 alpha2-globin, alpha1-globin, beta-globin, preferably
beta-globin, more preferably human beta-globin.
[0281] 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 present 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 .beta.-globin gene.
[0282] Preferably, RNA if delivered to, i.e. transfected into, a
cell, in particular a cell present in vivo, expresses the protein
or peptide it encodes.
[0283] The term "transfection" relates to the introduction of
nucleic acids, in particular RNA, into a cell. For purposes of the
present invention, the term "transfection" also includes the
introduction of a nucleic 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 vivo, 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 material is only transiently expressed. Since the nucleic
acid introduced in the transfection process is usually not
integrated into the nuclear genome, the foreign nucleic acid will
be diluted through mitosis or degraded. Cells allowing episomal
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.
[0284] The term "stability" of RNA relates to the "half-life" of
RNA. "Half-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 half-life 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.
[0285] In the context of the present invention, the term
"transcription" relates to a process, wherein the genetic code in a
DNA sequence is transcribed into RNA. Subsequently, the RNA may be
translated into protein. According to the present 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 present invention encompassed by the term
"vector".
[0286] The term "translation" according to the invention relates to
the process in the ribosomes of a cell by which a strand of
messenger RNA directs the assembly of a sequence of amino acids to
make a peptide or protein.
[0287] 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 particular to the production of peptides or proteins. 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".
[0288] "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 expression of
a certain protein, e.g., a tumor antigen. An increase in expression
refers to an increase 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.
[0289] The term "specifically expressed" means that a protein is
essentially only expressed in a specific tissue or organ. For
example, a tumor antigen specifically expressed 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 different 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
preferably to an approximately equal extent in lung and stomach,
said tumor antigen is specifically expressed in lung and
stomach.
[0290] According to the invention, the term "RNA encoding" means
that RNA, if present in the appropriate environment, preferably
within a cell, can be expressed to produce a protein or peptide it
encodes.
[0291] The term "peptide" according to the invention comprises
oligo- and polypeptides and refers to substances comprising 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, preferably 16 more, preferably 21 or
more and up to preferably 8, 10, 20, 30, 40 or 50, in particular
100 amino acids joined covalently by peptide bonds. The term
"protein" refers to large peptides, preferably to peptides with
more than 100 amino acid residues, but in general the terms
"peptides" and "proteins" are synonyms and are used interchangeably
herein.
[0292] 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.
[0293] 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 analogs
of given natural amino acids.
[0294] According to the invention, the term "cyclic peptide"
relates to a peptide or polypeptide chain which forms a ring. A
peptide can be cyclized in four different ways: head-to-tail
(C-terminus to N-terminus), head-to-side chain, side chain-to-tail
or side-chain-to-side-chain. Particularly preferred according to
the invention are peptides containing two or more residues
containing thiol groups such as cysteines which can form
intramolecular disulphide bridges giving cyclic peptides.
[0295] According to the invention, a peptide mimotope may be
covalently or non-covalently bound to one or more other compounds.
Such compounds include peptidic compound such as peptides and
proteins as well as non-peptidic compounds such as polyethylene
glycol (PEG).
[0296] In one embodiment, the peptide mimotopes described herein
are PEGylated. PEGylation is the process of covalent attachment of
polyethylene glycol (PEG) polymer chains to another molecule, such
as a peptide or protein. The covalent attachment of PEG can "mask"
the agent from the host's immune system (reduced immunogenicity and
antigenicity), and increase the hydrodynamic size (size in
solution) of the agent which prolongs its circulatory time by
reducing renal clearance. PEGylation can also provide water
solubility to hydrophobic drugs and proteins.
[0297] 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 specific
sequences resulting in sequences which are functionally equivalent
to said specific sequences, e.g. amino acid sequences exhibiting
properties identical or similar to those of the specific amino acid
sequences. One important property is to retain binding to a target
or to sustain effector functions. Preferably, a sequence which is a
variant with respect to a specific sequence, when it replaces the
specific sequence in an antibody retains binding of said antibody
to CLDN18.2 and preferably functions of said antibody as described
herein, e.g. CDC mediated lysis or ADCC mediated lysis.
[0298] It will be appreciated by those skilled in the art that in
particular the sequences of the CDR, hypervariable and variable
regions can be modified without losing the ability to bind
CLDN18.2. For example, 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 5, preferably
from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in
the CDRs. In addition, the hypervariable and variable regions may
be modified so that they show substantial homology with the regions
of antibodies specifically disclosed herein.
[0299] For the purposes of the present 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 protein are also called N-terminal and/or C-terminal truncation
variants.
[0300] Amino acid insertion variants comprise insertions of single
or two or more amino acids in a particular amino acid sequence. In
the case of amino acid sequence variants having an insertion, one
or more amino acid residues are inserted into a particular site in
an amino acid sequence, although random insertion with appropriate
screening of the resulting product is also possible.
[0301] Amino acid addition variants comprise amino- and/or
carboxy-terminal fusions of one or more amino acids, such as 1, 2,
3, 5, 10, 20, 30, 50, or more amino acids.
[0302] Amino acid deletion variants are characterized by the
removal of one or more amino acids from the sequence, such as by
removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The
deletions may be in any position of the protein.
[0303] Amino acid substitution 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., substitutions 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, proline, phenylalanine, methionine, tryptophan), and
uncharged polar (glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and
tyrosine are sometimes classified jointly as aromatic amino
acids.
[0304] Preferably the degree of similarity, preferably identity
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%, 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 embodiments, the degree of similarity or identity is
given for the entire length of the reference amino acid sequence.
The alignment for determining sequence 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.
[0305] "Sequence similarity" indicates the percentage of amino
acids that either are identical 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 sequences.
[0306] The term "percentage identity" is intended to denote a
percentage of amino acid residues which are identical between the
two sequences to be compared, obtained after the best alignment,
this percentage being purely statistical and the differences
between the two sequences 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 programs 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.).
[0307] The percentage identity is calculated by determining the
number of identical positions between the two sequences being
compared, dividing this number by the number of positions compared
and multiplying the result obtained by 100 so as to obtain the
percentage identity between these two sequences.
[0308] 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
invention to any cell which can be transfected with an exogenous
nucleic acid. Preferably, the cell when transfected with an
exogenous nucleic acid can express the nucleic acid.
[0309] "Reduce", "decrease" or "inhibit" as used herein means an
overall decrease or the ability to cause an overall decrease,
preferably of 5% or greater, 10% or greater, 20% or greater, more
preferably of 50% or greater, and most preferably of 75% or
greater, in the level, e.g. in the level of expression or in the
level of proliferation of cells.
[0310] 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%, at least 200%, at least 500%, at
least 1000%, at least 10000% or even more.
[0311] The peptide mimotopes described herein may be used in assays
for assaying the presence or amount of CLDN18.2 or CLDN18.2
antibodies, in particular the CLDN18.2 antibodies described herein.
Such assays may be carried out in a number of ways, including but
not limited to immunodetection, and include ELISA, in particular
peptide ELISA, competitive binding assays, and the like. In
general, in such assays an antibody or antibody fragment is used
that specifically binds the target peptide or protein and that is
directly or indirectly bound to a label that provides for
detection, e.g. indicator enzymes, radiolabels, fluorophores, or
paramagnetic particles. The methods of the invention allow
quantitative and/or qualitative evaluations, e.g., absolute and/or
relative evaluations, of CLDN18.2 or CLDN18.2 antibodies.
[0312] The term "enzyme-linked immunosorbent assay or ELISA", as
used herein, relates to a method for quantitatively or
semi-quantitatively determining protein concentrations from a
sample, e.g. blood plasma, serum or cell/tissue extracts, in a
multi-well plate format (usually 96-wells per plate). Broadly,
proteins in solution are adsorbed to ELISA plates. Antibodies
specific for the protein of interest may be used to probe the
plate. Background is minimized by optimizing blocking and washing
methods (as for IHC), and specificity is ensured via the presence
of positive and negative controls. Detection methods are usually
colorimetric or chemiluminescence based.
[0313] According to the invention, a label may function to: (i)
provide a detectable signal; (ii) interact with a second label to
modify the detectable signal provided by the first or second label,
e.g. FRET (Fluorescence Resonance Energy Transfer); (iii) affect
mobility, e.g. electrophoretic mobility, by charge, hydrophobicity,
shape, or other physical parameters, or (iv) provide a capture
moiety, e.g., affinity, antibody/antigen, or ionic complexation.
Suitable as label are structures, such as fluorescent labels,
luminescent labels, chromophore labels, radioisotopic labels,
isotopic labels, preferably stable isotopic labels, isobaric
labels, enzyme labels, particle labels, in particular metal
particle labels, magnetic particle labels, polymer particle labels,
small organic molecules such as biotin, ligands of receptors or
binding molecules such as cell adhesion proteins or lectins,
label-sequences comprising nucleic acids and/or amino acid residues
which can be detected by use of binding agents, etc. Labels
comprise, in a nonlimiting manner, barium sulfate, iocetamic acid,
iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine
diatrizoate, metrizamide, sodium tyropanoate and radio diagnostic,
including positron emitters such as fluorine-18 and carbon-11,
gamma emitters such as iodine-123, technetium-99m, iodine-131 and
indium-111, nuclides for nuclear magnetic resonance, such as
fluorine and gadolinium.
[0314] The term "sample", as used herein, includes any biological
sample which may be isolated from a patient and used for analysis
purposes. Said sample may be a body fluid sample, a tissue sample,
or a cell sample. For example, samples encompassed by the present
invention are tissue (e.g. section or explant) samples, single cell
samples, cell colony samples, cell culture samples, blood (e.g.
whole blood or blood fraction such as blood cell fraction, serum or
plasma) samples, urine samples, or samples from other peripheral
sources. Said samples may be mixed or pooled, e.g. a sample may be
a mixture of a blood sample and a urine sample. Said samples may be
provided by removing a body fluid, cell(s), cell colonies, an
explant, or a section from a patient, but may also be provided by
using a previously isolated sample. For example, a tissue sample
may be removed from a patient by conventional biopsy techniques or
a blood sample may be taken from a patient by conventional blood
collection techniques. The sample, e.g. tissue sample or blood
sample, may be obtained from a patient prior to initiation of the
therapeutic treatment, during the therapeutic treatment, and/or
after the therapeutic treatment.
[0315] In one embodiment, the sample is a body fluid sample. The
term "body fluid sample", as used herein, refers to any liquid
sample derived from the body of a patient. Said body fluid sample
may be a blood sample, urine sample, sputum sample, breast milk
sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample,
endolymph sample, perilymph sample, gastric juice sample, mucus
sample, peritoneal fluid sample, pleural fluid sample, saliva
sample, sebum (skin oil) sample, semen sample, sweat sample, tears
sample, vaginal secretion sample, or vomit sample including
components or fractions thereof. Said body fluid samples may be
mixed or pooled. Thus, a body fluid sample may be a mixture of a
blood and a urine sample or a mixture of a blood and cerebrospinal
fluid sample. Said body fluid sample may be provided by removing a
body liquid from a patient, but may also be provided by using
previously isolated body fluid sample material.
[0316] In one preferred embodiment, the sample is a whole blood
sample or a blood fraction sample such as a blood cell fraction,
blood serum, or blood plasma sample.
[0317] The agents such as peptide mimotopes described herein may be
administered in the form of any suitable pharmaceutical
composition.
[0318] The pharmaceutical compositions of the invention 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.
[0319] 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.
[0320] A pharmaceutical composition may comprise salts, buffer
substances, preservatives, carriers, diluents and/or excipients all
of which are preferably pharmaceutically acceptable. The term
"pharmaceutically acceptable" refers to the non-toxicity of a
material which does not interact with the action of the active
component of the pharmaceutical composition.
[0321] Salts which are not pharmaceutically acceptable may be used
for preparing pharmaceutically acceptable salts and are included in
the invention. Pharmaceutically acceptable salts of this kind
comprise in a non limiting way those prepared from the following
acids: hydrochloric, hydrobromic, sulfuric, 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.
[0322] 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 phosphoric acid in a salt.
[0323] Suitable preservatives for use in a pharmaceutical
composition include benzalkonium chloride, chlorobutanol, paraben
and thimerosal.
[0324] An injectable formulation may comprise a pharmaceutically
acceptable excipient such as Ringer Lactate.
[0325] 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.
[0326] Possible carrier substances for parenteral administration
are e.g. sterile water, Ringer, Ringer lactate, sterile sodium
chloride solution, polyalkylene glycols, hydrogenated naphthalenes
and, in particular, biocompatible lactide polymers,
lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene
copolymers.
[0327] The term "excipient" 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, flavouring agents, or
colorants.
[0328] The agents and compositions described herein are
administered in effective amounts. An "effective amount" refers to
the amount which achieves a desired reaction or a desired effect
alone or together with further doses. In the case of treatment of a
particular disease or of a particular condition, the desired
reaction preferably relates to inhibition of the course of the
disease. This comprises 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.
[0329] An effective amount of an agent or composition described
herein will depend on the condition 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.
[0330] 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 administering the agents and
compositions described herein. This includes disorders involving
cells characterized by an altered expression pattern of
CLDN18.2.
[0331] For example, in one embodiment, agents described herein can
be used to treat a patient with a cancer disease, e.g., a cancer
disease such as described herein characterized by the presence of
cancer cells expressing CLDN18.2.
[0332] 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.
[0333] The pharmaceutical composition of the invention may be
administered together with supplementing immunity-enhancing
substances such as one or more adjuvants and may comprise one or
more immunity-enhancing substances to further increase its
effectiveness, preferably to achieve a synergistic effect of
immunostimulation. The term "adjuvant" relates to compounds which
prolongs or enhances or accelerates an immune response. Various
mechanisms are possible in this respect, depending on the various
types of adjuvants. 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 context, 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, IL-8,
IL-9, IL-10, IL-12, INF.alpha., INF-.gamma., GM-CSF, LT-.alpha., or
growth factors, e.g. hGH. Further known adjuvants are aluminium
hydroxide, Freund's adjuvant or oil such as Montanide.RTM., most
preferred Montanide.RTM. ISA51. Lipopeptides, such as Pam3Cys, are
also suitable for use as adjuvants in the pharmaceutical
composition of the present invention.
[0334] The pharmaceutical composition can be administered locally
or systemically, preferably systemically.
[0335] 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 include 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.
[0336] According to the present invention, it is preferred that the
systemic administration 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 administration, intradermal administration or
intraarterial administration but is not limited thereto.
[0337] The present invention is further illustrated by the
following examples which are not be construed as limiting the scope
of the invention.
EXAMPLES
Example 1: Materials and Methods
[0338] Phage Display Selection:
[0339] IMAB362 binding peptides were identified by phage display
technology using a Ph.D..TM.-C7C phage display peptide library kit
(New England Biolabs) with a solution-phase panning protocol. For
the panning 50 .mu.l of Protein A Dynabeads (Invitrogen) were
washed once with 1 ml of TBST buffer (50 mM Tris-HCl (pH 7.5), 150
mM NaCl+0.1% Tween-20) and subsequently blocked with 1 ml blocking
buffer (3% BSA in TBS) for 1 h at 4.degree. C. Afterwards, the
beads were washed four times with 1 ml TBS-T buffer. A 100-fold
representation of the library (1E+11 phages) was incubated with 100
nM of the antibody for 20 min at RT. Antibody-bound phages were
captured with blocked Protein A beads by incubation for 15 min at
RT with slight shaking. After incubation, beads were washed ten
times with TBST to remove unbound phages. Elution was done by
subsequent incubation with 50 .mu.l of 100 mM triethylamine (for 6
min) and 50 .mu.l of 100 mM glycine-HCl, pH 2 (for 10 min). Eluates
were combined and immediately neutralized with 100 .mu.l 1 M
Tris-HCl, pH 7. Amplification of the neutralized phages was done
according to manufacturer's instructions by infection of log-phase
ER2738 E. coli cells. Amplified phages were purified from E. coli
supernatant also as described in the NEB manual by double PEG
precipitation. Purified phages were quantified photometrically and
used for subsequent panning rounds. Two additional screening rounds
were done with decreased antibody concentration (50 nM) and
increased washing stringency (washing with 0.5 Tween).
[0340] Single Phage Clone Analysis:
[0341] After the third screening round, individual phage clones
were generated and tested for target binding in a direct phage
ELISA. For this purpose, 1E+12 phages were coated to a 96 well
Maxisorp.TM. plate (Thermo Fisher Scientific) in coating buffer (50
mM sodium carbonate, pH 9.4) for 1 h at 37.degree. C. Eight
separate wells were coated per clone. The liquid was discarded and
wells were blocked overnight at 4.degree. C. with 300 .mu.l
blocking buffer (3% BSA in TBS buffer). After washing twice with
300 .mu.l TBS buffer per well, incubation with 500 nM IMAB362 or
control targets Rituximab or huIgG-Fc fragment (Merck Millipore;
100 .mu.l per well, diluted with 0.3% BSA in TBS) was done for 90
min at 4.degree. C. Wells were washed three times with TBS-T and
twice with TBS. Afterwards bound phages were detected with HRP
conjugated goat anti-human IgG antibody (Sigma-Aldrich, 1:5000
diluted with 0.3% BSA in TBS) and TMB substrate. For sequence
analysis phage DNA was isolated using the QIAprep Spin M13 kit
(Qiagen).
[0342] Peptide-Optimization with Peptide Microarrays:
[0343] Peptides were synthesized and printed on peptidemicroarray
slides essentially as described previously [Funkner, A. et al., J.
Mol. Biol. 2013, 425, 1340-1362]. In brief, the peptides were
synthesized using SPOT synthesis [Wenschuh, H. et al., Biopolymers
2000, 55, 188-206] cleaved from the solid support and cyclized (50%
DMSO, PBS buffer pH 7-8, RT, 16 hours). Subsequently, all peptides
were chemoselectively immobilized on functionalized glass slides as
described earlier [Funkner, A. et al., J. Mol. Biol. 2013, 425,
1340-1362]. Each peptide was deposited on the microarray in
triplicates.
[0344] The microarrays were incubated with 1.0 or 0.1 .mu.g/ml
IMAB362 in an HS 4800 microarray processing station (Tecan) for two
hours at 30.degree. C., followed by incubation with 1.0 .mu.g/ml
fluorescently labelled secondary antibody (Alexa647 anti-human Fc
antibody; Jackson Immuno Research). Washing steps were performed
prior every incubation step with 0.1% Tween-20 in 1.times.TBS.
After the final incubation step the microarrays were washed (0.05%
Tween-20 in 0.1.times.SSC) and dried in a stream of nitrogen. Each
microarray was scanned using a GenePix Autoloader 4200AL (Molecular
Devices, Pixel size: 10 .mu.m). Signal intensities were evaluated
using GenePix Pro 7.0 analysis software (Molecular Devices). For
each peptide, the mean of the three triplicates was calculated.
[0345] The lower limit of quantification (LLQ) value was defined as
LLQ=meanblank+6*SDblank. It was determined by plotting OD (405 nm)
values against IMAB362 concentration in the linear range (0-25
ng/ml) and analysis via linear regression.
[0346] Further evaluation of results was performed using the
statistical computing and graphics software R (Version 2.15.2,
www.r-project.org).
[0347] Synthesis of Purified Peptides:
[0348] The peptides shown in Table 2 were synthesized by standard
Fmoc-based solid-phase-synthesis protocols and purified. All target
peptides were obtained with a purity of >80%. See supporting
information for details.
[0349] Binding Analysis Using Biolayer Interferometry:
[0350] Binding analysis was performed using streptavidin (SA)
sensors on an Octet Red system (ForteBIO). 5 .mu.g/ml of
biotinylated peptides were used for loading (200 .mu.l per sensor).
To prevent unspecific binding 1.times. blocking buffer
(Sigma-Aldrich) was used in each step after loading. Remained
biotin binding sites on coated streptavidin sensors were blocked
using 100 .mu.g/ml biocytin (Sigma-Aldrich) for 5 minutes. For
binding mode analysis of different peptides 500 nM IMAB362 antibody
was used in the association step. For K.sub.D analysis the antibody
was used in a range of 15.625 to 500 nM (15.625, 31.25, 62.5, 125,
250, 500 nM) for each analyzed peptide. The following program was
used:
[0351] Baseline: 1000 rpm for 180 sec.
[0352] Load: 1000 rpm for 900 sec.
[0353] Baseline: 1000 rpm for 180 sec.
[0354] Blocking: 1000 rpm for 300 sec.
[0355] Association: 1000 rpm for 1200 sec.
[0356] Dissociation: 1000 rpm for 1200 sec.
[0357] Binding Analysis Using Peptide ELISA:
[0358] For the detection of IMAB362, the CLDN18.2 mimicking
peptides were immobilized on streptavidin plates (Nunc) in a
concentration of 0.75 .mu.g/ml in PBS buffer for 1 hour at
37.degree. C. Subsequently, the plate was washed three times with
washing buffer (0.01% Tween-20 in PBS buffer) before it was blocked
with 3% BSA in PBS overnight at 4.degree. C. To detect IMAB362, the
blocked plate was washed again three times with washing buffer
followed by the addition of IMAB362 and incubation for 30 min at
37.degree. C. After another washing step, it was incubated with an
alkaline phosphatase conjugated anti-human Fc antibody (Jackson
Immuno Research) for additional 30 min at 37.degree. C. Finally,
the plate was washed again before being incubated with 1.5 mg/ml of
the substrate PNPP (para-nitrophenyl phosphate) in appropriate
substrate buffer (1 M Diethanolamine, 0.05 mM MgCl2, 0.01% sodium
azide, pH 9.8) for 30 min at RT in the dark. 3 M KOH was used to
stop the enzymatic reaction. Absorbance was measured with a
microplate reader (Infinite M200, Tecan). For dual wavelength
analysis, 405 nm was chosen as measurement wavelength and 492 nm as
reference wavelength. Absorbance values were calculated by
subtraction of the measured values for the reference wavelength and
for the measurement wavelength. Calculation of apparent K.sub.D
values was done by fitting the data with Sigma Plot 10 using a
two-site saturation binding model.
[0359] To analyze the influence of serum, murine serum taken from
Balb/cJ mice or human serum type AB (Lonza) was spiked into the
ELISA samples and the assay was performed as described above.
[0360] In Vivo Pharmakokinetic Analysis:
[0361] IMAB362 (1 mg/mouse, approximately 50 mg/kg in PBS buffer),
Rituximab (1 mg/mouse, approximately 50 mg/kg in PBS buffer) or
only buffer (PBS) was intravenously injected into 11 weeks old
female Balb/cJ mice (6 mice per group). After 8, 24, 48, 72, 144,
168 and 192 hrs, blood samples were taken from two mice of each
group for serum preparation. The serum samples were to be measured
in the linear range of the assay (approximately 0.3-300 ng/ml).
Therefore, the serum samples were diluted 1:50000 with PBS buffer
containing 0.2% BSA (0.002% final serum content) and analyzed by
peptide ELISA as described above using the 2c mimotope peptide for
antibody capturing. An IMAB362 standard row in presence of murine
Balb/cJ control serum was assayed in parallel (0-25 ng/ml). The
standard raw data were fitted by linear regression to calculate the
antibody concentration from the mouse samples at the respective
timepoint. Before the pharmakokinetic analysis, a permission for
the animal experiment was obtained from the relevant local
authority.
[0362] Peptide Synthesis
[0363] Materials:
[0364] All solvents were used without further purification. Water
was demineralized with a demineralization system (Milli-Q Plus,
Millipore). The reagents were purchased from Advanced ChemTech
(Louisville, Ky., USA), Sigma-Aldrich (Deisenhofen, Germany),
Bachem (Basel, Schweiz), J. T. Baker (Phillipsburg, USA), NeoMPS
(Strassburg, Frankreich), GL Biochem (Shanghai, China) or Merck
Chemicals (Nottingham, UK) and used without further
purification.
[0365] Equipment:
[0366] RP-HPLC-MS-analysis was done with an Agilent Series 1200
System (2.times.1200 Series binary pump SL G1312B, 1200
thermostatted column compartment SL G1316B, 1200 series high perf.
autosampler SL G1367C, 1200 series micro-vacuum degasser G1379B,
1100/1200 automation interface G2254A, 1100/1200 wellplate handler
G2255A, VWD-UV detector G1314B, valve G1158A,) and coupled ESI-MS
(Agilent LC/MSD Quad SL system G1956B) on a Phenomenex Gemini-NX-3u
column at 30.degree. C. and a flow of 1.0 ml/min with a linear
gradient (5 95% B in 6 min, with A: 0.05% TFA in water and B: 0.05%
TFA in MeCN) and UV detection at a wavelength of 220 nm.
Preparative separations were done on a Dionex UltiMate 3000 HPLC
system with suitable gradients (solvent A: 0.05% TFA in water,
solvent B: 0.05% TFA in MeCN) and UV detection at 220 nm.
[0367] General Method for Peptide Synthesis:
[0368] The C-terminal amino acid (Fmoc-Gly-OH) was coupled to
chlorotrityl polystyrene resin (100-400 mg) to yield a loading of
approx. 0.3 mmol/g. Following Fmoc deprotection with a solution of
piperidine in DMF (15:85, 2.times.10 min), repeating cycles of
coupling of amino acids and Fmoc cleavage were performed until the
linear target peptides were assembled. For the coupling of the
amino acids the following reagents were employed: Fmoc-AA-OH (3.5
eq.), HOBt (1.0 eq.), DIC (3.5 eq.) in DMF for 75 min. For all
amino acids double couplings were performed. Biotin was coupled
with the following protocol: Biotin (5.0 eq.), HBTU (5.0 eq.),
DIPEA (10.0 eq.) in NMP for 16 hours. The peptides were cleaved off
the solid support with TFA/H2O/TIPS/EDT (90:3:4:3, RT, 3 hours) and
treated with MTBE at 0.degree. C. The suspension was centrifuged
and the supernatant removed to furnish the crude peptides. For
cyclization the peptides were dissolved in H2O/MeCN at a
concentration of 1 mg/mL. The solutions were brought to pH 7-7.5 by
addition of 2 M ammonia in H2O. After addition of DMSO (2%) the
solutions were stirred under an air atmosphere for 16-72 hours.
Upon completion of the cyclization, which was monitored by LC-MS,
the crude reaction mixture was acidified with TFA and lyophilized.
Purification was done by dissolution in MeCN/H.sub.2O and
fractionation by HPLC.
Example 2
[0369] Selection of IMAB362-Specific Peptides by Phage Display
[0370] For the identification of IMAB362-binding peptides phage
display was performed using a disulfide-constrained random 7mer M13
phage library (New England Biolabs). To ensure good accessibility
of the antibody a selection in solution protocol with protein-A
bead capture of binding phages was carried out. After three
consecutive screening rounds with increased stringency an
enrichment of target-specific phages could be observed, measured by
dilution plating of input and output phages and phage ELISA (data
not shown). Analysis of single clones randomly picked after the 3rd
screening round yielded several IMAB362 binders (FIG. 1A). The
phage ELISA suggests that the isolated peptides are specific for
the variable part of IMAB362, because a control antibody with
similar chimeric backbone (Rituximab) and a human IgG Fc fragment
were not recognized. DNA sequence analysis of selected clones
revealed a common YPG motif (FIG. 1B) in the middle of the
sequences. Three of the peptide sequences (sequence 1, 2, and 3;
FIG. 1B and Table 1) were selected for further binding and SAR
analysis and optimization via peptide microarrays.
TABLE-US-00014 TABLE 1 Peptide microarray incubation: results for
assay setup. % Affinity compared to hit peptide ACHLNYPGYCG
ACHYSYPGVCG ACHLGYPGRCG Peptide Structure.sup.[a]
Concentration.sup.[b] (Seq. 1) (Seq. 2) (Seq. 3)
Cyclopeptide-GGS-K(X) 1 100 100 100 X-Ttds-Cyclopeptide 1 95.9
(Peptide 98.3 (Peptide 107.8 (Peptide 1a) 2a) 3a)
X-Ttds-Cyclopeptide 0.1 2.6 22.9 11.6 X-Ttds-Linearpeptide 1 0 0
0.1 Shown are the results for nine different peptides (sequences
and structures indicated in table). Affinities were calculated from
fluorescence units as percentage of the fluorescence assay signal
of the hit peptides identified by phage display. .sup.[a]X is the
functional group being used for covalent attachment of the peptides
to the microarray. .sup.[b]Concentration of IMAB362 (in
.mu.g/ml).
[0371] SAR Analysis and Optimization of IMAB362-Specific Peptides
by Peptide Microarrays
[0372] Peptide microarrays were used for
structure-activity-relationship (SAR) analysis and optimization of
peptide binding affinity to IMAB362. For assay setup three variants
of each cyclic hit peptide (sequences 1, 2 and 3, Table 1) were
prepared differing in a) the immobilization site, b) the linker
nature and c) the degree of conformational restriction (cyclic vs.
linear). All peptides were chemically synthesized in a stepwise
fashion from the C- to the N-terminus by the high-throughput SPOT
synthesis approach [Wenschuh, H. et al., Biopolymers 2000, 55,
188-206] applying a cellulose membrane as solid support. After
cleavage and isolation, the peptides were cyclised through
disulfide formation between two internal Cys residues and
immobilized to the microarray. Covalent attachment to the
microarray was performed through a reactive moiety on either the N-
or C-terminus of the peptides allowing chemoselective and directed
immobilization through both ends of the peptides. Subsequently,
peptide loaded microarrays were incubated with 1.0 or 0.1 .mu.g/ml
IMAB362 followed by incubation with 1 .mu.g/ml fluorescently
labelled secondary antibody, scanning and data evaluation.
[0373] The results of the measurements for assay setup on the
peptide microarrays are shown in Table 1. The IMAB362 antibody
exhibits comparably strong binding to all parental (cyclic,
immobilized through C-terminus) peptides examined (Table 1, row 1).
The binding was independent from the immobilization site (C- vs.
N-terminal immobilization) (Table 1, row 2). As expected, reduction
of the IMAB concentration to 0.1 .mu.g/ml (Table 1, row 3) reduced
the assay signal. The final experiment (Table 1, row 4) showed that
cyclization is essential for binding as none of the linear peptides
furnished any signal in the microarray assay.
[0374] For peptide optimization substitutional analyses (exchange
of parental amino acids at each position by all proteinogenic amino
acids except Cys) of the three most promising peptides were
performed (peptides 1a, 2a and 3a, Table 1). In total 399 peptides
were synthesized, handled, cyclized and immobilized to microarrays
as described above. The results after incubation of the peptide
microarrays with IMAB362 are shown in FIG. 2. It became apparent
that substitution of several amino acids of the starting sequences
(sequences number 1, 2 and 3) by alternative amino acids resulted
in increased signal intensities. Several sequences showing highest
signal intensities were selected for re-synthesis, purification and
detailed binding analysis (FIG. 2, green boxes).
[0375] Detailed Binding Analysis of Optimized IMAB362-Specific
Peptides
[0376] The peptides displaying the highest binding affinities from
the peptide microarray experiments were synthesized by standard
Fmoc-based solid-phase-synthesis protocols and purified (HPLC
purity: >80%). The binding characteristics of each peptide were
determined. ELISA was used for assessment of the thermodynamic
behaviour, while biolayer interferometry (BLI) was applied to the
strongest binders for kinetic analysis (see also FIG. 5).
[0377] The results of the measurements shown in Table 2 indicate
that the affinity of each of the three parental peptides (peptides
1a, 2a and 3a) could be significantly improved. Correspondingly,
BLI data show the following improvements of affinity: peptide 1a
(810 nM) to peptide 1c (108 nM), peptide 2a (100 nM) to peptide 2c
(52 nM) and peptide 3a (320 nM) to peptide 3c (92 nM). In the ELISA
assay considerably improved IC50 values were measured: peptide 1a
(5.80 nM) to peptide 1c (0.26 nM), peptide 2a (4.44 nM) to peptide
2c (0.15 nM) and peptide 3a (1.61 nM) to peptide 3c (0.13 nM).
TABLE-US-00015 TABLE 2 Binding characteristics of parental and
maturated peptides. Apparent K.sub.D, Apparent K.sub.D, Peptide
Sequence.sup.[a] ELISA.sup.[b] [nM] BL.sup.[c] [nM] 1a
Biotin-Ttds-ACHLNYPGYCG-OH 5.80 810 1b Biotin-Ttds-ACHLNYPGWCG-OH
0.28 n.d. 1c Biotin-Ttds-ACHLGYPGYCG-OH 0.26 108 2a
Biotin-Ttds-ACHYSYPGVCG-OH 4.44 100 2b Biotin-Ttds-ACHYSYPGWCG-OH
2.12 n.d. 2c Biotin-Ttds-ACHYGYPGVCG-OH 0.15 52 3a
Biotin-Ttds-ACHLGYPGRCG-OH 1.61 320 3b Biotin-Ttds-ACHYGYPGRCG-OH
0.57 n.d. 3c Biotin-Ttds-ACHLGYPGWCG-OH 0.13 92 Shown are the
apparent binding constants for the parental and maturated IMAB362
binding peptides. .sup.[a]All peptides were cyclized by
Cys-Cys-disulfide bond formation. Ttds is a linker with the
following structure: 1,13-diamino-4,7,10-trioxatridecan-succinamic
acid. Amino acids that were exchanged compared to the original
sequence are underlined. .sup.[b]Shown values are the mean of at
least two individual measurements. .sup.[c]Measurement of at least
six different concentrations of IMAB362 over a time period of each
1200 sec. for association and dissociation. n.d. = not
determined.
[0378] For the development of an optimal detection system for
IMAB362 in biological samples the peptide 2 group (peptides 2a, 2b
and 2c, Table 2) was chosen for detailed peptide ELISA analysis,
because peptide 2c is the most affine variant when considering BLI
and ELISA data. For this purpose, the peptides were immobilized on
streptavidin coated 96-well plates and binding of IMAB362 and a
similar control antibody were measured by ELISA. FIG. 3A shows the
binding curves of the three peptides by comparison. To simulate the
situations of a pharmakokinetic study the most affine peptide 2c
was also analyzed in the presence of up to 20% human or mouse serum
(FIGS. 3B and 3C). It became evident that binding of IMAB362 to
peptide 2c is almost not affected by mouse serum components. The
binding curves looked similar when the antibody was diluted with
human serum although the background signal was slightly increased
especially at high serum concentrations.
[0379] Use of IMAB362-Specific Peptides for the Detection of
IMAB362 in Serum Samples from a Pharmacokinetic Study
[0380] To prove the applicability of the developed assay a
pharmacokinetic study was carried out. For this purpose, IMAB362
(approximately 50 mg/kg in PBS buffer), Rituximab (approximately 50
mg/kg in PBS buffer) or PBS buffer only were injected into Balb-cJ
mice. Blood samples were taken after different time points (8, 24,
48, 72, 144 and 192 hrs) and subsequently analyzed by peptide ELISA
using the 2c mimotope peptide for capturing of the antibody. FIG. 4
shows a decrease of the IMAB362 concentration in mice over time
starting from approximately 800 .mu.g/ml after 8 hours to
approximately 350 .mu.g/ml at the end of the study. All control
samples from the Rituximab and the PBS mice groups were negative,
which illustrates the high specificity of the developed assay.
Example 3: Influence of CLDN18.2 Mimotope on BiMAB Binding to NugC4
Target Cells and NugC4 Target Cell Lysis
[0381] Cell Culture
[0382] The human gastric cancer cell line NugC4 was derived from
the Japanese Collection of Research Bioresources and has been
stably transduced with the human CLDN18.2 gene in addition to a
stable transfection with the firefly luciferase gene. The cells
were cultivated in RPMI complete medium containing 10% FBS at
37.degree. C. in a 7.5% CO2 humidified incubator.
[0383] Preparation of Primary Human T-Cells
[0384] Human T-cells were freshly isolated from human blood from
healthy donors according to standard procedures (Current Protocols
in Immunology, 2012): briefly, blood was diluted with DPBS, layered
on Ficoll-Paque Plus (GE Healthcare Life Sciences) and centrifuged.
Peripheral blood mononuclear cells (PBMC) were collected from the
interphase, washed with cold DPBS supplemented with 2 mM EDTA and
counted. Human T-cells were subsequently separated by
magnetic-activated cell separation (MACS) from PBMCs by Pan T-Cell
Isolation Kit II (Miltenyi Biotec) according to the manufacturer's
guidelines.
[0385] Cytotoxicity Assay
[0386] To determine BiMAB-mediated lysis of CLDN18.2-expressing
NugC4 target cells by primary human T-cells, a luminescence-based
cytotoxicity assay was performed. Human T-cells were prepared as
described above. NugC4 cells stably expressing the luciferase gene
were used as target cells. 1.times.104 target cells were seeded per
well into white flat bottom 96-well plates. Human T cells were
added in an E:T ratio of 5:1. RPMI complete medium+10% FBS was used
to adjust the final volume to 100 .mu.l. Test samples and control
samples were plated at least in triplicates.
[0387] Cell culture microplates were incubated for 24 h at
37.degree. C., 5% CO2. Afterwards, 50 .mu.l of a aqueous solution
containing 1 mg/ml luciferin (BD Monolight, BD Biosciences) and 50
mM HEPES were added per well and plates subsequently incubated for
30 min in the dark at 37.degree. C. Luminescence arising from
oxidation of luciferin by luciferase expressing viable cells was
measured in a microplate-reader (Infinite M200, Tecan). Percentage
of specific target cell lysis was calculated by the following
formula: % specific lysis=[1-(luminescencetest
sample-Lmax)/(Lmin-Lmax)].times.100, whereas "L" indicates lysis.
Lmin refers to the minimum lysis in the absence of bi-scFv and Lmax
to the maximum lysis (equal to spontaneous luminescence counts) in
the absence of bi-scFv achieved by addition of Triton X-100 (2%
final concentration).
[0388] Cell Binding Assay
[0389] To assess the cancer target cell binding of a bispecific
single chain antibody specific for CLDN18.2 on cancer target cells,
2.times.105 NugC4 cells were incubated 30 min with 100, 1000 or
10000 ng/ml BiMAB in a final volume of 100 .mu.l at 4.degree. C.
Afterwards, cells were washed with 2 ml DPBS followed by staining
with 3.3 .mu.g/ml of an anti-His antibody (Dianova) for additional
30 min at 4.degree. C. before. Subsequently, cells were washed
again with 2 ml DPBS and stained with an allophycocyanin-conjugated
anti-mouse antibody (BD Biosciences) for 10 minutes at 4.degree. C.
Finally, cells were washed twice with 2 ml DPBS before the mean
fluorescence intensity was measured at FACS canto II (Becton
Dickinson).
[0390] FIG. 6 shows the influence of CLDN18.2 mimotope on BiMAB
binding to NugC4 target cells and NugC4 target cell lysis.
Sequence CWU 1
1
801261PRTHomo sapiens 1Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe
Val Val Ser Leu Ile1 5 10 15Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys
Met Asp Gln Trp Ser Thr 20 25 30Gln Asp Leu Tyr Asn Asn Pro Val Thr
Ala Val Phe Asn Tyr Gln Gly 35 40 45Leu Trp Arg Ser Cys Val Arg Glu
Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60Gly Tyr Phe Thr Leu Leu Gly
Leu Pro Ala Met Leu Gln Ala Val Arg65 70 75 80Ala Leu Met Ile Val
Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95Ser Ile Phe Ala
Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110Ala Lys
Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120
125Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val
130 135 140Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met
Gly Gly145 150 155 160Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe
Gly Ala Ala Leu Phe 165 170 175Val Gly Trp Val Ala Gly Gly Leu Thr
Leu Ile Gly Gly Val Met Met 180 185 190Cys Ile Ala Cys Arg Gly Leu
Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205Val Ser Tyr His Ala
Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220Phe Lys Ala
Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile225 230 235
240Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255Lys His Asp Tyr Val 2602118PRTArtificial
SequenceAntibody fragment 2Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile Asn Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Tyr Pro Ser Asp
Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser
Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ser
Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr
Leu Thr Val Ser Ser 1153113PRTArtificial SequenceAntibody fragment
3Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly1 5
10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn
Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro
Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
Val Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr Ser Tyr Pro Phe Thr Phe Gly
Ser Gly Thr Lys Leu Glu Ile 100 105 110Lys415PRTArtificial
SequenceLinker 4Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser1 5 10 15516PRTArtificial SequenceLinker 5Val Glu Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Asp1 5 10
1566PRTArtificial SequenceLinker 6Ser Gly Gly Gly Gly Ser1
575PRTArtificial SequenceLinker 7Gly Gly Gly Gly Ser1
5818PRTArtificial SequenceLinker 8Val Glu Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser Gly Gly1 5 10 15Val Asp920PRTArtificial
SequenceLinker 9Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly1 5 10 15Gly Gly Gly Ser 201025PRTArtificial
SequenceLinker 10Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
251118PRTArtificial SequenceLinker 11Gly Gly Gly Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly1 5 10 15Gly
Ser12467PRTArtificial SequenceDescription of artificial sequence
chimeric monoclonal antibody 12Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Arg 20 25 30Pro Gly Ala Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Trp Ile Asn
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Asn
Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn65 70 75 80Gln Lys Phe
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95Thr Ala
Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val 100 105
110Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly
115 120 125Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 130 135 140Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala145 150 155 160Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val 165 170 175Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys225 230
235 240Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 275 280 285Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345
350Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 370 375 380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460Pro
Gly Lys46513240PRTArtificial SequenceDescription of artificial
sequence chimeric monoclonal antibody 13Met Glu Ser Gln Thr Gln Val
Leu Met Ser Leu Leu Phe Trp Val Ser1 5 10 15Gly Thr Cys Gly Asp Ile
Val Met Thr Gln Ser Pro Ser Ser Leu Thr 20 25 30Val Thr Ala Gly Glu
Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45Leu Leu Asn Ser
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln 50 55 60Lys Pro Gly
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg65 70 75 80Glu
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90
95Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
100 105 110Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser
Gly Thr 115 120 125Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe 130 135 140Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys145 150 155 160Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val 165 170 175Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 180 185 190Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 195 200 205Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 210 215
220Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys225 230 235 240147PRTArtificial SequenceConsensus
sequenceMISC_FEATURE(1)..(1)Any amino acid, preferably an amino
acid selected from the group consisting of Gln, His, Tyr, Lys and
Met, more preferably an amino acid selected from the group
consisting of Gln, His and TyrMISC_FEATURE(2)..(2)Any amino acid,
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys, Tyr, Phe and Arg, more preferably an amino acid selected
from the group consisting of Pro, Leu, Lys and
TyrMISC_FEATURE(3)..(3)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Ala, Gly, Asn, Arg and
SerMISC_FEATURE(5)..(5)Any amino acid, preferably an amino acid
selected from the group consisting of Tyr, Pro and Arg, more
preferably an amino acid selected from the group consisting of Tyr
and ProMISC_FEATURE(6)..(6)Any amino acid, preferably an amino acid
selected from the group consisting of His, Gly, Lys and Arg, more
preferably an amino acid selected from the group consisting of His
and GlyMISC_FEATURE(7)..(7)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys, more preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg and
Val 14Xaa Xaa Xaa Tyr Xaa Xaa Xaa1 5159PRTArtificial
SequenceConsensus sequenceMISC_FEATURE(2)..(2)Any amino acid,
preferably an amino acid selected from the group consisting of Gln,
His, Tyr, Lys and Met, more preferably an amino acid selected from
the group consisting of Gln, His and TyrMISC_FEATURE(3)..(3)Any
amino acid, preferably an amino acid selected from the group
consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more preferably an
amino acid selected from the group consisting of Pro, Leu, Lys and
TyrMISC_FEATURE(4)..(4)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Ala, Gly, Asn, Arg and
SerMISC_FEATURE(6)..(6)Any amino acid, preferably an amino acid
selected from the group consisting of Tyr, Pro and Arg, more
preferably an amino acid selected from the group consisting of Tyr
and ProMISC_FEATURE(7)..(7)Any amino acid, preferably an amino acid
selected from the group consisting of His, Gly, Lys and Arg, more
preferably an amino acid selected from the group consisting of His
and GlyMISC_FEATURE(8)..(8)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys, more preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg and
Val 15Cys Xaa Xaa Xaa Tyr Xaa Xaa Xaa Cys1 51610PRTArtificial
SequenceConsensus sequenceMISC_FEATURE(3)..(3)Any amino acid,
preferably an amino acid selected from the group consisting of Gln,
His, Tyr, Lys and Met, more preferably an amino acid selected from
the group consisting of Gln, His and TyrMISC_FEATURE(4)..(4)Any
amino acid, preferably an amino acid selected from the group
consisting of Pro, Leu, Lys, Tyr, Phe and Arg, more preferably an
amino acid selected from the group consisting of Pro, Leu, Lys and
TyrMISC_FEATURE(5)..(5)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Ala, Gly, Asn, Arg and
SerMISC_FEATURE(7)..(7)Any amino acid, preferably an amino acid
selected from the group consisting of Tyr, Pro and Arg, more
preferably an amino acid selected from the group consisting of Tyr
and ProMISC_FEATURE(8)..(8)Any amino acid, preferably an amino acid
selected from the group consisting of His, Gly, Lys and Arg, more
preferably an amino acid selected from the group consisting of His
and GlyMISC_FEATURE(9)..(9)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys, more preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg and
Val 16Ala Cys Xaa Xaa Xaa Tyr Xaa Xaa Xaa Cys1 5
101711PRTArtificial SequenceConsensus
sequenceMISC_FEATURE(3)..(3)Any amino acid, preferably an amino
acid selected from the group consisting of Gln, His, Tyr, Lys and
Met, more preferably an amino acid selected from the group
consisting of Gln, His and TyrMISC_FEATURE(4)..(4)Any amino acid,
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys, Tyr, Phe and Arg, more preferably an amino acid selected
from the group consisting of Pro, Leu, Lys and
TyrMISC_FEATURE(5)..(5)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Ala, Gly, Asn, Arg and
SerMISC_FEATURE(7)..(7)Any amino acid, preferably an amino acid
selected from the group consisting of Tyr, Pro and Arg, more
preferably an amino acid selected from the group consisting of Tyr
and ProMISC_FEATURE(8)..(8)Any amino acid, preferably an amino acid
selected from the group consisting of His, Gly, Lys and Arg, more
preferably an amino acid selected from the group consisting of His
and GlyMISC_FEATURE(9)..(9)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys, more preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg and
Val 17Ala Cys Xaa Xaa Xaa Tyr Xaa Xaa Xaa Cys Gly1 5
10187PRTArtificial SequenceConsensus
sequenceMISC_FEATURE(1)..(1)Any amino acid, preferably an amino
acid selected from the group consisting of Gln, His, Tyr, Lys and
Met, more preferably an amino acid selected from the group
consisting of His and TyrMISC_FEATURE(2)..(2)Any amino acid,
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys, Tyr, Phe and Arg, more preferably an amino acid selected
from the group consisting of Leu, Lys, Tyr and
PheMISC_FEATURE(3)..(3)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Gly, Asn, Arg, Ser, Trp and
LysMISC_FEATURE(7)..(7)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys 18Xaa Xaa Xaa Tyr Pro Gly Xaa1
5199PRTArtificial SequenceConsensus sequenceMISC_FEATURE(2)..(2)Any
amino acid, preferably an amino acid selected from the group
consisting of Gln, His, Tyr, Lys and Met, more preferably an amino
acid selected from the group consisting of His and
TyrMISC_FEATURE(3)..(3)Any amino acid, preferably an amino acid
selected from the group consisting of Pro, Leu, Lys, Tyr, Phe and
Arg, more preferably an amino acid selected from the group
consisting of Leu, Lys, Tyr and PheMISC_FEATURE(4)..(4)Any amino
acid,
preferably an amino acid selected from the group consisting of Ala,
Gly, Asn, Arg, Ser, Lys, Trp, Phe and Tyr, more preferably an amino
acid selected from the group consisting of Gly, Asn, Arg, Ser, Trp
and LysMISC_FEATURE(8)..(8)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys 19Cys Xaa Xaa Xaa Tyr Pro Gly Xaa
Cys1 52010PRTArtificial SequenceConsensus
sequenceMISC_FEATURE(3)..(3)Any amino acid, preferably an amino
acid selected from the group consisting of Gln, His, Tyr, Lys and
Met, more preferably an amino acid selected from the group
consisting of His and TyrMISC_FEATURE(4)..(4)Any amino acid,
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys, Tyr, Phe and Arg, more preferably an amino acid selected
from the group consisting of Leu, Lys, Tyr and
PheMISC_FEATURE(5)..(5)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Gly, Asn, Arg, Ser, Trp and
LysMISC_FEATURE(9)..(9)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys 20Ala Cys Xaa Xaa Xaa Tyr Pro Gly
Xaa Cys1 5 102111PRTArtificial SequenceConsensus
sequenceMISC_FEATURE(3)..(3)Any amino acid, preferably an amino
acid selected from the group consisting of Gln, His, Tyr, Lys and
Met, more preferably an amino acid selected from the group
consisting of His and TyrMISC_FEATURE(4)..(4)Any amino acid,
preferably an amino acid selected from the group consisting of Pro,
Leu, Lys, Tyr, Phe and Arg, more preferably an amino acid selected
from the group consisting of Leu, Lys, Tyr and
PheMISC_FEATURE(5)..(5)Any amino acid, preferably an amino acid
selected from the group consisting of Ala, Gly, Asn, Arg, Ser, Lys,
Trp, Phe and Tyr, more preferably an amino acid selected from the
group consisting of Gly, Asn, Arg, Ser, Trp and
LysMISC_FEATURE(9)..(9)Any amino acid, preferably an amino acid
selected from the group consisting of Thr, Trp, Tyr, Glu, Arg, Val,
Ile, Leu, Met, Ala, Phe and Lys 21Ala Cys Xaa Xaa Xaa Tyr Pro Gly
Xaa Cys Gly1 5 10227PRTArtificial SequencePeptide mimotope 22Gln
Pro Ala Tyr Tyr His Thr1 5237PRTArtificial SequencePeptide mimotope
23His Leu Gly Tyr Pro Gly Arg1 5247PRTArtificial SequencePeptide
mimotope 24His Tyr Gly Tyr Pro Gly Arg1 5257PRTArtificial
SequencePeptide mimotope 25His Leu Gly Tyr Pro Gly Trp1
5267PRTArtificial SequencePeptide mimotope 26His Tyr Ser Tyr Pro
Gly Val1 5277PRTArtificial SequencePeptide mimotope 27His Tyr Gly
Tyr Pro Gly Val1 5287PRTArtificial SequencePeptide mimotope 28His
Tyr Ser Tyr Pro Gly Trp1 5297PRTArtificial SequencePeptide mimotope
29His Leu Arg Tyr Pro Gly Glu1 5307PRTArtificial SequencePeptide
mimotope 30His Tyr Arg Tyr Pro Gly Glu1 5317PRTArtificial
SequencePeptide mimotope 31His Leu Asn Tyr Pro Gly Tyr1
5327PRTArtificial SequencePeptide mimotope 32His Leu Gly Tyr Pro
Gly Tyr1 5337PRTArtificial SequencePeptide mimotope 33His Leu Asn
Tyr Pro Gly Trp1 5347PRTArtificial SequencePeptide mimotope 34Tyr
Lys Gly Tyr Pro Gly Tyr1 5357PRTArtificial SequencePeptide mimotope
35His Tyr Gly Tyr Pro Gly Trp1 5369PRTArtificial SequencePeptide
mimotope 36Cys Gln Pro Ala Tyr Tyr His Thr Cys1 5379PRTArtificial
SequencePeptide mimotope 37Cys His Leu Gly Tyr Pro Gly Arg Cys1
5389PRTArtificial SequencePeptide mimotope 38Cys His Tyr Gly Tyr
Pro Gly Arg Cys1 5399PRTArtificial SequencePeptide mimotope 39Cys
His Leu Gly Tyr Pro Gly Trp Cys1 5409PRTArtificial SequencePeptide
mimotope 40Cys His Tyr Ser Tyr Pro Gly Val Cys1 5419PRTArtificial
SequencePeptide mimotope 41Cys His Tyr Gly Tyr Pro Gly Val Cys1
5429PRTArtificial SequencePeptide mimotope 42Cys His Tyr Ser Tyr
Pro Gly Trp Cys1 5439PRTArtificial SequencePeptide mimotope 43Cys
His Leu Arg Tyr Pro Gly Glu Cys1 5449PRTArtificial SequencePeptide
mimotope 44Cys His Tyr Arg Tyr Pro Gly Glu Cys1 5459PRTArtificial
SequencePeptide mimotope 45Cys His Leu Asn Tyr Pro Gly Tyr Cys1
5469PRTArtificial SequencePeptide mimotope 46Cys His Leu Gly Tyr
Pro Gly Tyr Cys1 5479PRTArtificial SequencePeptide mimotope 47Cys
His Leu Asn Tyr Pro Gly Trp Cys1 5489PRTArtificial SequencePeptide
mimotope 48Cys Tyr Lys Gly Tyr Pro Gly Tyr Cys1 5499PRTArtificial
SequencePeptide mimotope 49Cys His Tyr Gly Tyr Pro Gly Trp Cys1
55011PRTArtificial SequencePeptide mimotope 50Ala Cys Gln Pro Ala
Tyr Tyr His Thr Cys Gly1 5 105111PRTArtificial SequencePeptide
mimotope 51Ala Cys His Leu Gly Tyr Pro Gly Arg Cys Gly1 5
105211PRTArtificial SequencePeptide mimotope 52Ala Cys His Tyr Gly
Tyr Pro Gly Arg Cys Gly1 5 105311PRTArtificial SequencePeptide
mimotope 53Ala Cys His Leu Gly Tyr Pro Gly Trp Cys Gly1 5
105411PRTArtificial SequencePeptide mimotope 54Ala Cys His Tyr Ser
Tyr Pro Gly Val Cys Gly1 5 105511PRTArtificial SequencePeptide
mimotope 55Ala Cys His Tyr Gly Tyr Pro Gly Val Cys Gly1 5
105611PRTArtificial SequencePeptide mimotope 56Ala Cys His Tyr Ser
Tyr Pro Gly Trp Cys Gly1 5 105711PRTArtificial SequencePeptide
mimotope 57Ala Cys His Leu Arg Tyr Pro Gly Glu Cys Gly1 5
105811PRTArtificial SequencePeptide mimotope 58Ala Cys His Tyr Arg
Tyr Pro Gly Glu Cys Gly1 5 105911PRTArtificial SequencePeptide
mimotope 59Ala Cys His Leu Asn Tyr Pro Gly Tyr Cys Gly1 5
106011PRTArtificial SequencePeptide mimotope 60Ala Cys His Leu Gly
Tyr Pro Gly Tyr Cys Gly1 5 106111PRTArtificial SequencePeptide
mimotope 61Ala Cys His Leu Asn Tyr Pro Gly Trp Cys Gly1 5
106211PRTArtificial SequencePeptide mimotope 62Ala Cys Tyr Lys Gly
Tyr Pro Gly Tyr Cys Gly1 5 106311PRTArtificial SequencePeptide
mimotope 63Ala Cys His Tyr Gly Tyr Pro Gly Trp Cys Gly1 5
10645PRTArtificial SequencePeptide mimotope 64Tyr Leu His Pro Asp1
56512PRTArtificial SequencePeptide mimotope 65Thr Pro Tyr His His
Pro Asp Phe Pro Tyr Trp Phe1 5 10667PRTArtificial SequencePeptide
mimotope 66Tyr Leu His Pro Asp Tyr Pro1 5677PRTArtificial
SequencePeptide mimotope 67Tyr Leu His Pro Asp Val Met1
56835PRTArtificial SequencePeptide mimotope 68Pro Arg Cys Lys Ser
Glu Gly Pro His His Pro Asp Tyr Pro Asp Cys1 5 10 15Arg Arg Asp Ser
Asp Cys Asn Gly Glu Cys Ile Cys Arg Gly Asn Gly 20 25 30Tyr Cys Gly
356910PRTArtificial SequencePeptide mimotope 69Ala Cys Arg His Pro
Asp His Leu Asp Cys1 5 107010PRTArtificial SequencePeptide mimotope
70Ala Cys His Glu Thr His His Pro Asp Cys1 5 107112PRTArtificial
SequencePeptide mimotope 71Ser Phe Arg Asp Met Asn Tyr Ser Asp Tyr
Phe Met1 5 10727PRTArtificial SequencePeptide mimotope 72His Ile
Leu Pro Leu Tyr Pro1 5737PRTArtificial SequencePeptide mimotope
73Ser Pro Tyr Met Pro Met Gln1 57417PRTArtificial SequencePeptide
mimotope 74Asp Arg Cys Trp Leu Glu Gln Trp Pro Cys Arg Arg Asp Ser
Asp Ile1 5 10 15Pro7535PRTArtificial SequencePeptide mimotope 75Gln
Thr Cys Asp His Asp Thr Arg His Pro Thr Gly Asp Asp Leu Cys1 5 10
15Arg Arg Asp Ser Asp Cys Gly Gly Asn Cys Ile Cys Arg Gly Asn Gly
20 25 30Tyr Cys Gly 357614PRTArtificial SequencePeptide mimotope
76Ala Cys His Leu Gly Tyr Pro Gly Arg Cys Gly Gly Gly Ser1 5
107714PRTArtificial SequencePeptide mimotope 77Ala Cys His Tyr Ser
Tyr Pro Gly Val Cys Gly Gly Gly Ser1 5 107814PRTArtificial
SequencePeptide mimotope 78Ala Cys His Leu Asn Tyr Pro Gly Tyr Cys
Gly Gly Gly Ser1 5 107914PRTArtificial SequencePeptide mimotope
79Ala Cys His Leu Arg Tyr Pro Gly Glu Cys Gly Gly Gly Ser1 5
108014PRTArtificial SequencePeptide mimotope 80Ala Cys Tyr Lys Gly
Tyr Pro Gly Tyr Cys Gly Gly Gly Ser1 5 10
* * * * *
References