POLYPEPTIDES TETRAMERES SE LIANT A HER2

HER2-BINDING TETRAMERIC POLYPEPTIDES

Abrégé français

L'invention concerne un polypeptide tétramère comprenant une première chaîne polypeptidique comprenant un premier domaine de liaison à l'antigène VL et un premier domaine constant CL, une seconde chaîne polypeptidique comprenant un premier domaine de liaison à l'antigène VH, un premier domaine constant CH1, un premier domaine constant CH2 et un premier domaine constant CH3, un premier ligand se liant à un épitope de HER2 D4 lié à l'extrémité N-terminale dudit premier domaine de liaison à l'antigène VL ou dudit premier domaine de liaison d'antigène VH par un premier lieur d'acide aminé interdomaine, une troisième chaîne polypeptidique comprenant un second domaine de liaison à l'antigène VL et un second domaine constant CL, une quatrième chaîne polypeptidique comprenant un second domaine de liaison à l'antigène VH, un second domaine constant CH1, un second domaine constant CH2 et un second domaine constant CH3 et un troisième ligand se liant à un épitope de HER2 D4 lié à l'extrémité N-terminale dudit second domaine de liaison à l'antigène VL ou dudit second domaine de liaison à l'antigène VH par un second lieur d'acide aminé interdomaine, les domaines de liaison à l'antigène VL et les domaines de liaison à l'antigène VH constituant ensemble un second ligand et un quatrième ligand se liant à un épitope de HER2 D1. L'invention concerne en outre un polypeptide tétramère destiné à être utilisé dans un méthode de prévention ou de traitement d'une maladie néoplasique maligne, un acide nucléique isolé et une cellule hôte pour l'expression du polypeptide ainsi qu'un procédé permettant d'obtenir le polypeptide.

Abrégé anglais

The invention relates to a tetrameric polypeptide comprising a first polypeptide chain comprising a first VL antigen binding domain and a first CL constant domain, a second polypeptide chain comprising a first VH antigen binding domain, a first CH1 constant domain, a first CH2 constant domain and a first CH3 constant domain, a first ligand binding to a HER2 D4 epitope linked to the N-terminus of said first VL antigen binding domain or said first VH antigen binding domain by a first interdomain amino acid linker, a third polypeptide chain comprising a second VL antigen binding domain and a second CL constant domain, a fourth polypeptide chain comprising a second VH antigen binding domain, a second CH1 constant domain, a second CH2 constant domain and a second CH3 constant domain and a third ligand binding to a HER2 D4 epitope linked to the N-terminus of said second VL antigen binding domain or said second VH antigen binding domain by a second interdomain amino acid linker, wherein the VL antigen binding domains and the VH antigen binding domains together constitute a second ligand and a fourth ligand binding to a HER2 D1 epitope. The invention further relates to the tetrameric polypeptide for use in a method for the prevention or treatment of a malignant neoplastic disease, an isolated nucleic acid and a host cell for expression of the polypeptide and a method for obtaining the polypeptide.

Revendications

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Claims
1. A tetrameric polypeptide comprising or consisting of
a. a first polypeptide chain comprising a first VL antigen binding domain and
a
first CL constant domain,
b. a second polypeptide chain comprising a first VH antigen binding domain, a
first CH1 constant domain, a first CH2 constant domain and a first CH3
constant domain,
c. a first ligand that specifically binds to a HER2 D4 epitope, wherein said
first
ligand is comprised in said first polypeptide chain and linked to the N-
terminus
of said first VL antigen binding domain by a first interdomain amino acid
linker,
or said first ligand is comprised in said second polypeptide chain and linked
to
the N-terminus of said first VH antigen binding domain by a first interdomain
amino acid linker,
d. wherein the first VL antigen binding domain of the first polypeptide chain
and
the first VH antigen binding domain of the second polypeptide chain together
constitute a second ligand, particularly a Fab domain, that specifically binds
to
a HER2 D1 epitope,
e. a third polypeptide chain comprising a second VL antigen binding domain and
a second CL constant domain,
f. a fourth polypeptide chain comprising a second VH antigen binding domain, a
second CH1 constant domain, a second CH2 constant domain and a second
CH3 constant domain,
g. a third ligand that specifically binds to a HER2 D4 epitope, wherein said
third
ligand is comprised in said third polypeptide chain and linked to the N-
terminus of said second VL antigen binding domain by a second interdomain
amino acid linker, or said third ligand is comprised in said fourth
polypeptide
chain and linked to the N-terminus of said second VH antigen binding domain
by a second interdomain amino acid linker,
h. wherein the second VL antigen binding domain of the third polypeptide chain
and the second VH antigen binding domain of the fourth polypeptide chain
together constitute a fourth ligand, particularly an Fab domain, that
specifically
binds to a HER2 D1 epitope.
2. The polypeptide according to claim 1, wherein
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- said first polypeptide chain and said third polypeptide chain and/or said
second
polypeptide chain and said fourth polypeptide chain are characterized by a
sequence
identity with each other of 70% or more, particularly 80% or more, more
particularly
90% or more, even more particularly 95% or more, wherein most particularly
said first
polypeptide chain and said third polypeptide chain and/or said second
polypeptide
chain and said fourth polypeptide chain are identical and/or wherein
- said first ligand and said third ligand are characterized by a sequence
identity with
each other of 70% or more, particularly 80% or more, more particularly 90% or
more,
even more particularly 95% or more, wherein most particularly said first
ligand and
said third ligand are identical.
3. The tetrameric polypeptide according to any one of the preceding claims,
wherein
said first CH2 constant domain and said first CH3 constant domain of said
second
polypeptide chain interact with, particularly are covalently linked with, said
second
CH2 constant domain and said second CH3 constant domain of said fourth
polypeptide chain, such that a tetrameric polypeptide is formed.
4. The tetrameric polypeptide according to any one of the preceding claims,
wherein
said second polypeptide chain comprises a first hinge region between said
first CH1
constant domain and said first CH2 constant domain, and said fourth
polypeptide
chain comprises a second hinge region between said second CH1 constant domain
and said second CH2 constant domain, wherein said first hinge region and said
second hinge region mediate complex formation between said second polypeptide
chain and said fourth polypeptide chain, particularly by at least one
disulphide bond,
such that a tetrameric polypeptide is formed.
5. The polypeptide according to any one of the preceding claims, wherein said
first
ligand and/or said third ligand comprises or consists of a single-chain
variable
fragment polypeptide chain comprising an scFy heavy chain, an scFy linker
chain,
and an scFy light chain, wherein particularly
a. said scFv heavy chain of said first ligand and/or said third ligand
comprises a
peptide sequence characterized by a sequence identity of 70 % or more, more
particularly 80 % or more, even more particularly 90 % or more, even more
particularly 95 % or more, with a peptide sequence selected from SEQ ID No.
15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID
No. 53, SEQ ID No. 54 and SEQ ID No. 80, wherein most particularly said
scFy heavy chain of said first ligand and/or said third ligand comprises a
peptide sequence identical to a peptide sequence selected from SEQ ID No.
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15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID
No. 53, SEQ ID No. 54 and SEQ ID No. 80, and
b. said scFv light chain of said first ligand and/or said third ligand
comprises a
peptide sequence characterized by a sequence identity of 70 % or more, more
particularly 80 % or more, even more particularly 90 % or more, even more
particularly 95 % or more, with a peptide sequence selected from SEQ ID No.
14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43 and SEQ
ID No. 81, wherein most particularly said scFv light chain of said first
ligand
and/or said third ligand comprises a peptide sequence identical to a peptide
sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ
ID No. 26, SEQ ID No. 43 and SEQ ID No. 81.
6. The polypeptide according to claim 5, wherein said scFv linker chain
comprises a
peptide sequence characterized by a sequence identity of 70 % or more,
particularly
80 % or more, more particularly 90 % or more, even more particularly 95 % or
more,
with SEQ ID No. 16, wherein most particularly said scFv linker chain comprises
a
peptide sequence identical to SEQ ID No. 16.
7. The polypeptide according to any one of the preceding claims, wherein
a. said first polypeptide chain and/or said third polypeptide chain comprises
a
peptide sequence characterized by a sequence identity of 70 % or more,
particularly 80 % or more, more particularly 90 % or more, even more
particularly 95 % or more, with a peptide sequence selected from SEQ ID No.
18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID
No. 41, SEQ ID No. 50 and SEQ ID 76, wherein most particularly said first
polypeptide chain and/or said third polypeptide chain comprises a peptide
sequence identical to a peptide sequence selected from SEQ ID No. 18, SEQ
ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41,
SEQ ID No. 50 and SEQ ID No. 76, and
b. said second polypeptide chain and/or said fourth polypeptide chain
comprises
a peptide sequence characterized by a sequence identity of 70 % or more,
particularly 80 % or more, more particularly 90 % or more, even more
particularly 95 % or more, with a peptide sequence selected from SEQ ID No.
19, SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID
No. 40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77,
wherein most particularly said second polypeptide chain and/or said fourth
polypeptide chain comprises a peptide sequence identical to a peptide
sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27, SEQ
73

ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51,
SEQ ID No. 52 and SEQ ID 77.
8. The polypeptide according to any one of the preceding claims, wherein
a. said first polypeptide chain and/or said third polypeptide chain comprises
a
peptide sequence characterized by a sequence identity of 70 % or more,
particularly 80 % or more, more particularly 90 % or more, even more
particularly 95 % or more, with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38
and SEQ ID No. 78, wherein most particularly said first polypeptide chain
and/or said third polypeptide chain comprises a peptide sequence identical to
SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, and
b. said second polypeptide chain and/or said fourth polypeptide chain
comprises
a peptide sequence characterized by a sequence identity of 70 % or more,
particularly 80 % or more, more particularly 90 % or more, even more
particularly 95 % or more, with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35
and SEQ ID No. 79, wherein most particularly said second polypeptide chain
and/or said fourth polypeptide chain comprises a peptide sequence identical
to SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79.
9. The polypeptide according to any one of the preceding claims, wherein
a. said first polypeptide chain and/or said third polypeptide chain is
characterized
by a sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or more, even more particularly 95 % or more, with SEQ ID
No. 1, wherein most particularly said first polypeptide chain and/or said
third
polypeptide chain is identical to SEQ ID No. 1, and
b. said second polypeptide chain and/or said fourth polypeptide chain is
characterized by a sequence identity of 70 % or more, particularly 80 % or
more, more particularly 90 % or more, even more particularly 95 % or more,
with SEQ ID No. 2, wherein most particularly said second polypeptide chain
and/or said fourth polypeptide chain is identical to SEQ ID No. 2.
10. The polypeptide according to any one of the preceding claims, wherein
a. said first polypeptide chain and/or said third polypeptide chain is
characterized
by a sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or more, even more particularly 95 % or more, with SEQ ID
No. 3, wherein most particularly said first polypeptide chain and/or said
third
polypeptide chain is identical to SEQ ID No. 3, and
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b. said second polypeptide chain and/or said fourth polypeptide chain is
characterized by a sequence identity of 70 % or more, particularly 80 % or
more, more particularly 90 % or more, even more particularly 95 % or more,
with SEQ ID No. 4, wherein most particularly said second polypeptide chain
and/or said fourth polypeptide chain is identical to SEQ ID No. 4.
11. The polypeptide according to any one of the preceding claims, wherein said
first
interdomain amino acid linker and/or said second interdomain amino acid linker
consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 amino
acids, and wherein said first interdomain amino acid linker and/or said second
interdomain amino acid linker comprises or consists of amino acids G, A, J, S,
T, P,
C, V, M and E, particularly wherein said first interdomain amino acid linker
and/or said
second interdomain amino acid linker comprises or consists of amino acids G,
S, A
and T, more particularly wherein said first interdomain amino acid linker
and/or said
second interdomain amino acid linker is characterized by an amino acid
sequence
(GGGGS)n, with n being 1, 2, 3, 4 or 5 or said first interdomain amino acid
linker
and/or said second interdomain amino acid linker comprises or consists of a
peptide
sequence selected from one of SEQ ID No. 17, SEQ ID 55 to SQ ID 69 and SEQ ID
82 to SEQ ID 91 or a functional equivalent peptide sequence characterized by a
sequence identity of at least 70%.
12. The polypeptide according to any one of the claims 1 to 11 for use in a
method for the
prevention or treatment of a malignant neoplastic disease associated with
expression
of HER2.
13. An isolated nucleic acid encoding at least one of the first polypeptide
chain, the
second polypeptide chain, the third polypeptide chain and the fourth
polypeptide
chain of the polypeptide according to any one of the claims 1 to 11.
14. A host cell which is adapted to produce at least one of the first
polypeptide chain, the
second polypeptide chain, the third polypeptide chain and the fourth
polypeptide
chain of the polypeptide according to any one of the claims 1 to 11, wherein
particularly the host cell comprises the isolated nucleic acid according to
claim 13,
such that the host cell is able to produce at least one of the first
polypeptide chain,
the second polypeptide chain, the third polypeptide chain and the fourth
polypeptide
chain of the polypeptide according to any one of the claims 1 to 11.
15. A method for obtaining the polypeptide according to any one of the claims
1 to 11,
wherein the method comprises culturing the host cell according to claim 14, so
that at
least one of the first polypeptide chain, the second polypeptide chain, the
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polypeptide chain and the fourth polypeptide chain of the polypeptide
according to
any one of the claims 1 to 11 is produced.
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Description

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HER2-binding tetrameric polypeptides
The present invention relates to a tetrameric polypeptide having two binding
sites to HER2
epitope D1 and two binding sites to HER2 epitope D4.
Background of the Invention
The members of the HER family of receptor tyrosine kinases are important
mediators of cell
growth, differentiation, migration and survival. The receptor family includes
four distinct
members including epidermal growth factor receptor (EGFR, ErbB1, or HER1),
HER2 (ErbB2
or p185<neu>), HER3 (ErbB3) and HER4 (ErbB4). The members of the EGFR family
are
closely related single-chain modular glycoproteins with an extracellular
ligand binding region,
a single transmembrane domain and an intracellular tyrosine kinase, followed
by specific
phosphorylation sites which are important for the docking of downstream
signaling proteins.
The extracellular regions of the HER receptor family contain two homologous
ligand binding
domains (domains 1 and 3) and two cysteine-rich domains (domains 2 and 4),
which are
important for receptor dimerization. In the absence of a ligand, HER receptors
normally exist
as inactive monomers, known as the "tethered" structure, which is
characterized by close
interaction of domain 2 and 4. Ligand binding to the extracellular domain
initiates a
conformational rearrangement, exposing the dimerization domains 2 and 4.
Therefore,
binding of growth factors to HER receptors induces conformational changes that
allow
receptor dimerization. After extracellular receptor dimerization,
transmembrane helices
switch to an active conformation that enables the intracellular kinase domains
to trans-auto-
phosphorylate each other. This phosphorylation event allows the recruitment of
specific
downstream signaling proteins.
Epidermal Growth factor receptor 1, (EGFR), has been causally implicated in
human
malignancy. In particular, increased expression of EGFR has been observed in
breast,
bladder, lung, head, neck and stomach cancer as well as glioblastomas.
Human epidermal growth factor receptor 2 (HER2, also known as ErbB2 or Neu;
UniProtKB/Swiss-Prot No. P04626) consists of 1233 amino acids and is
structurally similar to
EGFR, with an extracellular domain consisting of four subdomains 1-4, a
transmembrane
domain, a juxtamembrane domain, an intracellular cytoplasmic tyrosine kinase
and a
regulatory C-terminal region. The structure of HER2's extracellular region is
different in
important aspects from the other EGF receptors, however. In the other EGF
receptors, in a
non-activated state, domain 2 binds to domain 4. Upon binding to domains 1 and
3, the
activating growth factor (ligand) selects and stabilizes a conformation that
allows a
dimerization arm to extend from domain 2 to interact with an ErbB dimer
partner. HER2, on
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the other hand, has a fixed conformation that resembles the ligand-activated
state of the
other receptor members: the domain 2-4 interaction is absent and the
dimerization loop in
domain 2 is continuously exposed. HER2 is activated via formation of
heterodimeric
complexes with other ErbB family members and thereby indirectly regulated by
EGFR and
HER3 ligands. HER2 is the preferred heterodimerization partner of the three
other ErbB
receptors, enhancing the affinity of the other ErbB receptors for their
ligands by slowing down
the rate of ligand-receptor complex dissociation, whereby HER2 enhances and
prolongs
signaling.
An excess of HER2 on the cell surface causes transformation of epithelial
cells from multiple
tissues. Amplification of the human homolog of the neu gene (also known as
HER2) is
observed in breast and ovarian cancers and correlates with a poor prognosis
(US 4,968,603). Overexpression of HER2 has also been observed in other
carcinomas
including carcinomas of the stomach, endometrium, salivary gland, lung,
kidney, colon,
thyroid, pancreas and bladder.
Antibodies targeting HER2
Drebin and colleagues have raised antibodies against the rat neu gene product,
p185<neu>disclosed in U56,733,752.
Hudziak et al. (1989, Mol. Cell. Biol. 9(3), 1165-1172) describe the
generation of a panel of
HER2 antibodies which were characterized using the human breast tumor cell
line SkBr-3.
Using a cell proliferation assay, maximum inhibition was obtained with an
antibody called
4D5. The antibody 4D5 was further found to sensitize HER2-overexpressing
breast tumor
cell lines to the cytotoxic effects of TNF-[alpha]; see also US 5,677,171. A
recombinant
humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2,
trastuzumab or HERCEPTIN; US 5,821,337) is clinically active in patients with
HER2-
overexpressing metastatic breast cancers that have received extensive prior
anti-cancer
therapy. Herceptin is approved in combination with chemotherapy for use in
patients with
HER2-positive metastatic stomach (gastric) cancer.
Herceptin is widely used for the treatment of patients with early as well as
metastatic breast
cancer whose tumors overexpress HER2 protein and/or have HER2 gene
amplification. The
treatment of breast cancer patients with Herceptin/trastuzumab is, for
example,
recommended and now routine for patients having HER2-positive disease; see
US 2002/0064785, US 2003/0170234A1, U52003/0134344 and US 2003/0147884. The
prior
art thus focuses on the eligibility of breast cancer patients for
trastuzumab/Herceptin therapy
based on a high HER2 protein expression level (e.g. defined as HER2(3+) by
immunohistochemistry (IHC)). HER2-positive disease in breast cancer is defined
to be
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present if a high HER2 (protein) expression level is detected by
immunohistochemical
methods (e.g. HER2 (+++) or as HER2 gene amplification (e.g. a HER2 gene copy
number
higher than 4 copies of the HER2 gene per tumor cell) or both, found in
samples obtained
from the patients such as breast tissue biopsies or breast tissue resections
or in tissue
derived from metastatic sites. One frequently applied method for detecting
HER2
overexpression and amplification at the gene level is fluorescence in situ
hybridization
(FISH), which is also described in US 2003/0152987.
Pertuzumab, a humanized antibody, is the first of a new class of agents known
as HER
dimerization inhibitors (HDIs). Pertuzumab binds to HER2 at its dimerization
domain, thereby
inhibiting its ability to form active heterodimer receptor complexes, thus
blocking the
downstream signal cascade that ultimately results in cell growth and division.
Pertuzumab is
directed against the extracellular domain 2 of HER2. In contrast to
trastuzumab, which acts
by binding to domain 4 of HER2, pertuzumab is a HER dimerization inhibitor
which inhibits
dimerization of HER2 with HER3 and the other members of the EGFR receptor
family in the
.. presence of the respective activating ligands. By blocking complex
formation, pertuzumab
prevents the growth-stimulatory effects and cell survival signals activated by
ligands of
HER1, HER3 and HER4. Pertuzumab has been approved by the FDA under the name
Perjeta for treatment in combination with trastuzumab and docetaxel for
patients with HER2-
positive metastatic breast cancer, who have not received prior anti-HER2
therapy or
chemotherapy for metastatic disease. Pertuzumab is a fully humanized
recombinant
monoclonal antibody based on the human IgG1([kappa]) framework sequences.
Patent
publications concerning pertuzumab and selection of patients for therapy
therewith include:
U520060073143A1; U520030086924; U520040013667A1, and U520040106161A1.
For trastuzumab, while known to show clinical benefits in terms of e.g.
prolonged survival in
combination with chemotherapy compared to chemotherapy alone, a majority of
HER2
positive breast cancer patients were nevertheless found to be non-responders
(45% overall
response rate for Herceptin + chemotherapy vs. 29% for chemotherapy alone).
Thus, while monoclonal antibody therapy directed against HER2 has been shown
to provide
improved treatment in e.g. metastatic breast cancers that overexpress HER2,
there is still
considerable room for improvement.
Non-antibody scaffolds targeting HER2
Alternative targeting proteins have been proposed recently, which are more
diverse in
molecular structure than human immunoglobulin-derived antibody fragments and
antibody-
derived constructs and formats, and thus allow additional molecular formats by
creating
heterodimeric and multimeric assemblies, leading to new biological functions.
A number of
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such targeting proteins have been described (reviewed in (Binz et al., 2005,
Nat. Biotech, 23,
1257-1268)). Non-limiting examples of such targeting proteins are camelid
antibodies,
protein scaffolds derived from protein A domains (termed "Affibodies",
Affibody AB),
tendamistat (an alpha-amylase inhibitor, a 74 amino acid beta-sheet protein
from
Streptomyces tendae), fibronectin, lipocalin ("Anticalins", Pieris), T-cell
receptors, ankyrins
(designed ankyrin repeat proteins termed "DARPins", Univ. Zurich and Molecular
Partners;
see US20120142611), A-domains of several receptors ("Avimers", Avidia) and PDZ
domains, fibronectin domains (FN3) ("Adnectins", Adnexus), consensus
fibronectin domains
("Centyrins", Centyrex/Johnson&Johnson) and Ubiquitin ("Affilins", SOIL
Proteins) and
knottins (Moore and Cochrane, 2012, Methods in Enzymology 503, 223-251 and
references
cited therein).
From these proteins, multimeric and multispecific assemblies can be
constructed (Caravella
and Lugovskoy, 2010, Current Opinions in Chemical Biology, 14, 520-528;
Vanlandschoot et
al., 2011, Antiviral Research 92, 389-407; Lofblom et al., 2011, Current
Opinion in
Biotechnology, 22, 843-848, Boersma et al., 2011, Curr. Opin. Biotechnol., 22,
849-857). It is
also possible to fuse these and other peptidic domains to antibodies to create
so-called
Zybodies (Zyngenia Inc., Gaithersburg, MD).
All of these scaffolds, with different inherent properties, have in common
that they can be
directed to bind specific epitopes, by using selection technologies well known
to practitioners
in the field (Binz et al., ibid.).
For example, the different individual domains of HER2 can be individually
expressed in
insect cells, using a baculovirus expression system, as demonstrated for
domain 1 and
domain 4 (Frei et al., 2012, Nat Biotechnol., 30, 997-1001). Thereby, it is
guaranteed that
binders selected will be directed towards the domain of interest. The HER2
domains can
then be biotinylated as previously described (Zahnd et al., 2006, J. Biol.
Chem. 281(46),
35167-75), and thus be immobilized on streptavidin-coated magnetic beads or on
microtiter
plates coated with streptavidin or neutravidin (Steiner et al., 2008, J. Mol.
Biol. 382, 1211-
1227; Zahnd et al., 2007, J. Mol. Biol. 369, 1015-1028.). The HER2 domains so
immobilized
can then serve as targets for diverse protein libraries in either phage
display or ribosome
display format. A large variety of different antibody libraries has been
published (Mondon P.
et al., 2008, Frontiers in Bioscience. 13, 1117-1129) and the technology of
selecting binding
antibodies is well known to the practitioners of the field. Phage display is a
suitable format for
antibody fragments (Fab fragments, scFy fragments or single domain antibodies
s)
(Hoogenboom, 2005, Nature Biotechnology., 23(9), 1105-1116) and any other
scaffold that
.. contain disulfide bonds, but it can also be used for scaffolds not
containing disulfide bonds
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(e.g., Steiner et al., 2008, J. Mol. Biol., 382, 1211-1227) (Rentero et al.,
2011, Chimia.,
65(11), 843-5, Skerra A., 2007, Current Opinion in Biotechnology., 18(4), 295-
304). Similarly,
ribosome display can be used for antibody fragments (Hanes et al., 2000, Nat.
Biotechnol.,
18, 1287-1292) and for other scaffolds (Zahnd et al., 2007, Nat. Methods, 4,
269-279; Zahnd
et al., 2007, J. Mol. Biol., 369, 1015-28.). A third powerful technology is
yeast display
(Pepper et al., 2008, Combinatorial Chemistry & High Throughput Screening,
11(2), 127-
134). In this case a library of the binding protein of interest is displayed
on the surface of
yeast, and the respective domain of HER2 is either directly labeled with a
fluorescent dye or
its his tag is detected with an anti-histag antibody, which is in turn
detected with a secondary
antibody. Such methods are well known to the practitioners in the field (Boder
et al., 2000,
Methods in Enzymology, 328, 430-44).
Another possibility of engineering represents the connection of those binders
to create
bispecific or higher multivalent binding molecules. Such connection can be
achieved
genetically by fusions of two or more of these binding molecules or chemically
by
crosslinking separately expressed molecules, or by adding a dimerization
domain include
separate dependent claims for each or any combination thereof (see, e.g.
Stefan et al., 2011,
J. Mol. Biol., 413, 826-843; Boersma et al., 2011, J. Biol. Chem., 286, 41273-
41285).
A bispecific anti-HER2 camelidae antibody construct (Bispecific Nanobody) is
shown in
U520110059090. The document relates to a bispecific molecule that
simultaneously targets
HER2 at the extracellular domain 2, defined by competition with pertuzumab,
and domain 4,
defined by competition with trastuzumab. This molecule has been described to
exhibit
stronger anti-proliferative activity than trastuzumab (Herceptin) in a direct
comparison in an in
vitro cell culture model using the cell line SkBr3.
Due to the absence of any known HER2-specific ligand, current HER2 targeting
strategies
aim to block the dimerization of the receptor by binding to the interaction
interface. Today's
knowledge of HER2 receptor dimerization is mostly based on the crystal
structure of the
ligand-bound form of the EGFR homodimer, which is broadly accepted as the
active mode of
all EGF receptor family members (Garret et al., 2002, Cell, 110, 763-773). The
two EGFR
molecules show a back-to-back interaction. Extending these findings to HER2
and its
possible interaction with other members of the EGFR family, one interaction
interface is
present on domain 2 of the extracellular part of HER2. Pertuzumab binds to
domain 2 and is
indeed known to block receptor interaction at this interface. Another known
interaction is
present on domain 4 of the extracellular part of HER2. This interaction
interface is
presumably blocked by trastuzumab. Yet both antibodies, trastuzumab and
pertuzumab,
even when simultaneously applied, are not able to block all HER2 interactions
to
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completeness. The interaction of the extracellular part and the kinase domain
of HER2 are
thought to be linked in such a way as to allow some residual interactions even
in the
trastuzumab- and pertuzumab-blocked state, which is in accordance with crystal
structure
data (Lu et al., 2010, Mol. Cell. Biol., 22, 5432-5443). The bispecific ligand
mentioned above
that binds both epitopes (pertuzumab and trastuzumab) simultaneously (US
2011/0059090)
reduces the cell growth in a cell culture model by approx. 50%, in comparison
to a reduction
of about 40% effected by trastuzumab. This same effect, however, can also be
achieved by
treating with the mixture of trastuzumab and pertuzumab.
Patent application WO 2014/060365 describes bispecific HER2-targeting agents
comprising
a first polypeptide ligand that binds to HER2 extracellular domain 1 (D1
epitope) and a
second polypeptide ligand that binds to HER2 extracellular domain 4 (D4
epitope), wherein
the first and the second polypeptide ligand are separated by linker. The most
active ones of
these biparatopic binding agents bind predominantly to two separate HER2
molecules in an
intermolecular binding mode. Thereby these biparatopic bivalent binding agents
crosslink
HER2_ECD1 of one HER2 and HER2_ECD4 of the other HER2 molecule via these
paratopes and a preferentially short peptide linker, which favors inter- over
intramolecular
binding. The bivalent binding mode of the biparatopic binding agents will
induce a
polymerization of HER2 receptors at the cell surface, which are consequently
not able to
form productive HER2/HER3 or HER2/EGFR heterodimers or HER2/HER2 homodimers
(Tamaskovic et al., 2016, Jost et al., 2013). This consequently leads to an
inhibition of HER2
and HER3 phosphorylation in HER2-overexpressing cancer cells, which
subsequently leads
to cessation of proliferative and anti-apoptotic signaling from HER2/HER3
receptor and
finally induction of cell death by apoptosis. However, these biparatopic
binding agents do not
affect the total HER2 receptor expression levels (Tamaskovic et al., 2016).
The total HER2
receptor expression remains comparably high, which may cause an incomplete
inactivation
of HER2 receptors. Furthermore, these constructs do not show the desired
pharmacokinetic
properties, which are required for systemic applications. Desired functions
such as long
serum half-life in the blood stream or mechanisms of protein recycling via
binding to the
FcRn receptor are not implemented in these constructs. Furthermore, these
biparatopic
constructs do not harness the antibody effector functions such as complement-
dependent
cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).
These effector
functions may be beneficial to increase further the anti-tumor activity of the
biparatopic
binding agents in vivo. Finally, these biparatopic binding agents are
potentially prone to
induce an immune response, because they have not been further engineered to
avoid T-cell
epitopes. This may lead to significant reduction of tolerability and serum
level at repeated
dosage in immunocompetent patients.
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Furthermore, a tetravalent biparatopic HER2-targeting antibody-drug conjugate
comprising a
fusion of Trastuzumab and 39S antibody variable sequences binding to the D4
and D2
epitopes of HER2 has been described (Li et al., 2016, Cancer Cell, 29, 117-
129). This
conjugate was commercialized under the name MEDI4276 by Medimmune and was
tested in
a clinical phase 1/2 trial (NCT02576548). However, this biparatopic IgG fusion
construct in
its unarmed form, i.e., without the fusion of the Tubulysine toxic payload, is
not sufficient to
induce inhibition of cancer cell proliferation. On the contrary, the
unconjugated version of this
IgG fusion molecule induces activation of cancer cell proliferation at high
concentrations in
HER2-overexpressing cancer cell models. This may be caused by the binding to
HER2_ECD2 and HER2_ECD4 in a manner that increases the formation of signaling-
competent HER2 homo- and heterodimers. On the other hand, the unconjugated IgG
fusion
protein version of this tetravalent biparatopic HER2-targeting antibody can
induce
downregulation of HER2 receptor expression (Li et al., 2016, Cancer Cell, 29,
117-129). In
summary, the tetravalent scFv_4D5-IgG_39S fusion protein downregulates HER2
expression, yet it shows no inhibition of HER2 signaling and instead leads to
activation of
cancer cell proliferation in specific HER2-overexpressing models. This also
shows that
downregulation of HER2 and cancer cell growth inhibition are not simply
linked. Clearly,
inhibition of HER2-dependent signaling pathways of a HER2-targeting agent
would be
essential for clinical applications. For example, trastuzumab (Herceptin),
does block signaling
of HER3 and does show significant reduction of cell proliferation in HER2-
overexpressing
cancers that do not have a PI3K-pathway activating mutation.
Based on the above-mentioned state of the art, the objective of the present
invention is to
provide means and methods to provide a HER2-targeting agent which is improved
in view of
the above-stated disadvantages of the prior art, in particular to provide a
HER2-targeting
agent displaying improved HER2 signalling inhibition, downregulation of
expression,
polymerization and clustering, inhibition of receptor diffusion, degradation,
and/or inhibition of
recycling, in the absence of additional small molecule toxins bound to the
HER2 targeting
agent. This objective is attained by the subject-matter of the independent
claims of the
present specification.
Summary of the Invention
The invention relates to a tetrameric polypeptide comprising or consisting of
- a first polypeptide chain comprising in N to C orientation a first
VL antigen binding
domain and a first CL constant domain,
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- a second polypeptide chain comprising in N to C orientation a first VH
antigen binding
domain, a first CH1 constant domain, a first CH2 constant domain and a first
CH3
constant domain,
- a first ligand that specifically binds to a HER2 D4 epitope, wherein the
first ligand is
comprised in the first polypeptide chain and linked to the N-terminus of the
first VL
antigen binding domain by a first interdomain amino acid linker, or the first
ligand is
comprised in the second polypeptide chain and linked to the N-terminus of the
first
VH antigen binding domain by a first interdomain amino acid linker,
- wherein the first VL antigen binding domain of the first polypeptide
chain and the first
VH antigen binding domain of the second polypeptide chain together constitute
a
second ligand, particularly a Fab domain, that specifically binds to a HER2 D1
epitope,
- a third polypeptide chain comprising in N to C orientation a second VL
antigen
binding domain and a second CL constant domain,
- a fourth polypeptide chain comprising in N to C orientation a second VH
antigen
binding domain, a second CH1 constant domain, a second CH2 constant domain and
a second CH3 constant domain,
- a third ligand that specifically binds to a HER2 D4 epitope, wherein the
third ligand is
comprised in the third polypeptide chain and linked to the N-terminus of the
second
VL antigen binding domain by a second interdomain amino acid linker, or the
third
ligand is comprised in the fourth polypeptide chain and linked to the N-
terminus of the
second VH antigen binding domain by a second interdomain amino acid linker,
- wherein the second VL antigen binding domain of the third polypeptide
chain and the
second VH antigen binding domain of the fourth polypeptide chain together
constitute
a fourth ligand, particularly an Fab domain, that specifically binds to a HER2
D1
epitope.
The tetrameric polypeptide according to the invention thus comprises two times
two different
HER2-binding paratopes, in other words, is tetravalent. The polypeptide is
biparatopic,
because it contains binding sites to two distinct HER2 epitopes, namely D1 and
D4 on a
single molecule.
Surprisingly, the polypeptide displays superior HER2 inactivation compared to
conventional
antibodies and divalent biparatopic polypeptides (comprised of a total of two
binding
paratopes) and has additional effects on cell growth and proliferation,
apoptosis and other
forms of cell death, HER2 internalization and HER2 recycling inhibition, HER2
expression
downregulation and HER2 degradation, HER2 crosslinking, inhibition of HER2
dimerization,
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and decrease of HER2 receptor surface mobility. Furthermore, the increased
molecular size
excludes renal filtration, and FcRn-mediated recycling will increase the
pharmacokinetic
properties. Finally, the presence of the Fc part also allows antibody-
dependent cell-mediated
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to occur.
Lastly, all
sequences are those of human antibodies, and thus the construct consists of
non-
immunogenic sequences. In summary, the tetrameric polypeptides according to
the present
invention are promising candidates for systemic therapy of HER2-expressing
cancer.
In another embodiment, the present invention relates to a pharmaceutical
composition
comprising at least one of the compounds of the present invention or a
pharmaceutically
acceptable salt thereof and at least one pharmaceutically acceptable carrier,
diluent or
excipient.
The invention further relates to the tetrameric polypeptide for use in a
method for the
prevention or treatment of a malignant neoplastic disease, an isolated nucleic
acid encoding
the polypeptide, a host cell for producing the polypeptide and a method for
obtaining the
polypeptide.
Detailed Description of the Invention
Terms and definitions
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art (e.g., in
cell culture,
molecular genetics, nucleic acid chemistry, hybridization techniques and
biochemistry).
Standard techniques are used for molecular, genetic and biochemical methods
(see
generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed.
(1989) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short
Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and
chemical
methods.
The term "a subject comprises an object" in the context of the present
specification includes
discrete embodiments where the subject consists of the object, in other words
where the
term comprise is synonymous with "consist of". In other discrete embodiments,
the object is
one of several different comprised in the object.
The term ligand in the context of the present specification relates to a
region of a polypeptide
binding to a target, particularly HER2.
The term interdomain amino acid linker in the context of the present
specification relates to a
polypeptide linker covalently connecting the C-terminus of a first polypeptide
domain to the
N-terminus of a second polypeptide domain.
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The term epitope in the context of the present specification relates to a
region of an antigen
molecule to which an antibody binds.
The term Fab domain in the context of the present specification relates to an
antibody
molecule comprising a first chain consisting of a VL domain C-terminally
(covalently) linked
to a CL domain and a second chain consisting of a VH domain C- terminally
(covalently)
linked to a CH1 domain, wherein the CL domain and the CH1 domain are linked by
a
disulfide bond.
The term VL antigen binding domain in the context of the present specification
relates to the
variable domain of the light chain of an antibody, particularly of an
immunoglobulin G light
chain.
The term VH antigen binding domain in the context of the present specification
relates to the
variable domain of the heavy chain of an antibody, particularly of an
immunoglobulin G light
chain.
The term CL constant domain in the context of the present specification
relates to the
constant domain of the light chain of an antibody, particularly of an
immunoglobulin G heavy
chain.
The term CH1 constant domain in the context of the present specification
relates to the first
constant domain of the heavy chain of an antibody, particularly of an
immunoglobulin G
heavy chain.
The term CH2 constant domain in the context of the present specification
relates to the
second constant domain of the heavy chain of an antibody, particularly of an
immunoglobulin
G heavy chain.
The term CH3 constant domain in the context of the present specification
relates to the third
constant domain of the heavy chain of an antibody, particularly of an
immunoglobulin G
heavy chain.
The term single-chain variable fragment in the context of the present
specification relates to
a VH antigen binding domain (also termed scFv heavy chain) covalently linked
to a VL
antigen binding domain (also termed scFv light chain) by a polypeptide linker
(scFv linker
chain).
In the present specification, the term positive, when used in the context of
expression of a
marker, refers to expression of an antigen assayed by a fluorescently labelled
antibody,
wherein the label's fluorescence on the structure (for example, a cell)
referred to as "positive"
is at least 30% higher 30 %), particularly 50`)/0 or E30%, in median
fluorescence intensity
in comparison to staining with an isotype-matched fluorescently labelled
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not specifically bind to the same target. Such expression of a marker is
indicated by a
superscript "plus" (i), following the name of the marker, e.g. CD4+.
In the present specification, the term negative, when used in the context of
expression of a
marker, refers to expression of an antigen assayed by a fluorescently labelled
antibody,
wherein the median fluorescence intensity is less than 30% higher,
particularly less than 15%
higher, than the median fluorescence intensity of an isotype-matched antibody
which does
not specifically bind the same target. Such expression of a marker is
indicated by a
superscript minus (-), following the name of the marker, e.g. CD127-.
High expression of a marker, for example high expression of 0D25, refers to
the expression
level of such marker in a clearly distinguishable cell population that is
detected by FACS
showing the highest fluorescence intensity per cell compared to the other
populations
characterized by a lower fluorescence intensity per cell. A high expression is
indicated by
superscript "high" or "hi" following the name of the marker, e.g. CD25hIgh.
The term "is
expressed highly" refers to the same feature.
Low expression of a marker, for example low expression of 0D25, refers to the
expression
level of such marker in a clearly distinguishable cell population that is
detected by FACS
showing the lowest fluorescence intensity per cell compared to the other
populations
characterized by higher fluorescence intensity per cell. A low expression is
indicated by
superscript "low" or "lo" following the name of the marker, e.g. CD2510w. The
term "is
expressed lowly" refers to the same feature.
The expression of a marker may be assayed via techniques such as fluorescence
microscopy, flow cytometry, ELISPOT, ELISA or multiplex analyses.
The term polypeptide in the context of the present specification relates to a
molecule
consisting of 50 or more amino acids that form a linear chain wherein the
amino acids are
connected by peptide bonds. The amino acid sequence of a polypeptide may
represent the
amino acid sequence of a whole (as found physiologically) protein or fragments
thereof. The
term "polypeptides" and "protein" are used interchangeably herein and include
proteins and
fragments thereof. Polypeptides are disclosed herein as amino acid residue
sequences.
The term peptide in the context of the present specification relates to a
molecule consisting
of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly
8 to 15amino
acids, that form a linear chain wherein the amino acids are connected by
peptide bonds.
Amino acid residue sequences are given from amino to carboxyl terminus.
Capital letters for
sequence positions refer to L-amino acids in the one-letter code (Stryer,
Biochemistry, 3rd ed.
p. 21). Lower case letters for amino acid sequence positions refer to the
corresponding D- or
(2R)-amino acids. Sequences are written left to right in the direction from
the amino to the
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carboxy terminus. In accordance with standard nomenclature, amino acid residue
sequences
are denominated by either a three letter or a single letter code as indicated
as follows:
Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp,
D), Cysteine
(Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G),
Histidine (His, H),
.. lsoleucine (Ile, l), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met,
M), Phenylalanine
(Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan
(Trp, W), Tyrosine
(Tyr, Y), and Valine (Val, V). J is leucine or isoleucine.
The term gene refers to a polynucleotide containing at least one open reading
frame (ORF)
that is capable of encoding a particular polypeptide or protein after being
transcribed and
translated. A polynucleotide sequence can be used to identify larger fragments
or full-length
coding sequences of the gene with which they are associated. Methods of
isolating larger
fragment sequences are known to those of skill in the art.
The terms gene expression or alternatively gene product refer to the processes
- and
products thereof - of nucleic acids (RNA) or amino acids (e.g., peptide or
polypeptide) being
.. generated when a gene is transcribed and translated.
As used herein, expression refers to the process by which DNA is transcribed
into mRNA
and/or the process by which the transcribed mRNA is subsequently translated
into peptides,
polypeptides or proteins. If the polynucleotide is derived from genomic DNA,
expression may
include splicing of the mRNA in a eukaryotic cell. Expression may be assayed
both on the
level of transcription and translation, in other words mRNA and/or protein
product.
Sequences similar or homologous (e.g., at least about 70% sequence identity)
to the
sequences disclosed herein are also part of the invention. In some
embodiments, the
sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%,
92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the
sequence identity
can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or higher. Alternatively, substantial identity exists when the nucleic
acid segments will
hybridize under selective hybridization conditions (e.g., very high stringency
hybridization
conditions), to the complement of the strand. The nucleic acids may be present
in whole
cells, in a cell lysate, or in a partially purified or substantially pure
form.
Calculations of "homology" or "sequence identity" or "similarity" between two
sequences (the
terms are used interchangeably herein) are performed as follows. The sequences
are
aligned for optimal comparison purposes (e.g., gaps can be introduced in one
or both of a
first and a second amino acid or nucleic acid sequence for optimal alignment
and non-
homologous sequences can be disregarded for comparison purposes). In a
particular
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embodiment, the length of a reference sequence aligned for comparison purposes
is at least
30%, particularly at least 40%, more particularly at least 50%, even more
particularly at least
60%, and even more particularly at least 70%, 80%, 90%, 100% of the length of
the
reference sequence. The amino acid residues or nucleotides at corresponding
amino acid
positions or nucleotide positions are then compared. When a position in the
first sequence is
occupied by the same amino acid residue or nucleotide as the corresponding
position in the
second sequence, then the molecules are identical at that position (as used
herein amino
acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid
"identity"). The
percent identity between the two sequences is a function of the number of
identical positions
shared by the sequences, taking into account the number of gaps, and the
length of each
gap, which need to be introduced for optimal alignment of the two sequences.
In the case of
circularly related proteins, the sequence of one of the partners needs to be
appropriately split
and aligned in two sections to achieve optimal alignment of the functionally
equivalent
residues necessary to calculate the percent identity.
In the context of the present specification, the terms sequence identity and
percentage of
sequence identity refer to the values determined by comparing two aligned
sequences.
Methods for alignment of sequences for comparison are well-known in the art.
Alignment of
sequences for comparison may be conducted by the local homology algorithm of
Smith and
Waterman, 1981, Adv. Appl. Math., 2, 482, by the global alignment algorithm of
Needleman
and Wunsch, 1970, J. Mol. Biol., 48, 443, by the search for similarity method
of Pearson and
Lipman, 1988, Proc. Nat. Acad. Sci., 85, 2444 or by computerized
implementations of these
algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA
and
TFASTA. Software for performing BLAST analyses is publicly available, e.g.,
through the
National Center for Biotechnology-Information
(http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm
that uses
the default settings: Expect threshold: 10; Word size: 3; Max matches in a
query range: 0;
Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional
adjustments:
Conditional compositional score matrix adjustment. One such example for
comparison of
nucleic acid sequences is the BLASTN algorithm that uses the default settings:
Expect
threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch
Scores: 1.-
2; Gap costs: Linear. Unless stated otherwise, sequence identity values
provided herein refer
to the value obtained using the BLAST suite of programs (Altschul et al.,
1990, J. Mol. Biol.,
215, 403-410) using the above identified default parameters for protein and
nucleic acid
comparison, respectively.
In the context of the present specification, the term antibody refers to whole
antibodies
including but not limited to immunoglobulin type G (IgG), type A (IgA), type D
(IgD), type E
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(IgE) or type M (IgM), any antigen binding fragment or single chains thereof
and related or
derived constructs. A whole antibody is a glycoprotein comprising at least two
heavy (H)
chains and two light (L) chains inter-connected by disulfide bonds. Each heavy
chain is
comprised of a heavy chain variable region (VH) and a heavy chain constant
region (CH). The
heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
Each light
chain is comprised of a light chain variable region (abbreviated herein as VL)
and a light
chain constant region (CO. The light chain constant region is comprised of one
domain, CL.
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 of the classical complement system.
Similarly, the
term encompasses a so-called nanobody or single domain antibody, an antibody
fragment
consisting of a single monomeric variable antibody domain.
In the context of the present specification, the term humanized antibody
refers to an antibody
originally produced by immune cells of a non-human species, the protein
sequences of which
have been modified to increase their similarity to antibody variants produced
naturally in
humans. The term humanized antibody as used herein includes antibodies in
which CDR
sequences derived from the germline of another mammalian species, such as a
mouse, have
been grafted onto human framework sequences. Additional framework region
modifications
may be made within the human framework sequences as well as within the CDR
sequences
derived from the germline of another mammalian species.
The term antibody-like molecule in the context of the present specification
refers to a
molecule capable of specific binding to another molecule or target with high
affinity / a
Kd 5 10E-8 mo1/1. An antibody-like molecule binds to its target similarly to
the specific
binding of an antibody. The term antibody-like molecule encompasses a repeat
protein, such
as a designed ankyrin repeat protein (Molecular Partners, Zurich), an
engineered antibody
mimetic proteins exhibiting highly specific and high-affinity target protein
binding (see US
2012/142611, US 2016/250341, US 2016/075767 and US 2015/368302, all of which
are
incorporated herein by reference). The term antibody-like molecule further
encompasses, but
is not limited to, a polypeptide derived from armadillo repeat proteins, a
polypeptide derived
from leucine-rich repeat proteins and a polypeptide derived from
tetratricopeptide repeat
proteins.
The term antibody-like molecule further encompasses a specifically binding
polypeptide
derived from
- a protein A domain,
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- fibronectin domain FN3,
- consensus fibronectin domains,
- a lipocalins (see Skerra, 2000, Biochim. Biophys. Acta, 1482(1-2), 337-
50),
- a polypeptide derived from a Zinc finger protein (see Kwan et al., 2003,
Structure,
11(7), 803-813),
- Src homology domain 2 (5H2) or Src homology domain 3 (5H3),
- a PDZ domain,
- gamma-crystallin,
- ubiquitin,
- a cysteine knot polypeptide or a knottin,
- cystatin,
- Sac7d,
- a triple helix coiled coil (also known as alphabodies),
- a Kunitz domain or a Kunitz-type protease inhibitor and
- a carbohydrate binding module 32-2.
The term protein A domains derived polypeptide refers to a molecule that is a
derivative of
protein A and is capable of specifically binding the Fc region and the Fab
region of
immunoglobulins.
The term armadillo repeat protein refers to a polypeptide comprising at least
one armadillo
repeat, wherein an armadillo repeat is characterized by a pair of alpha
helices that form a
hairpin structure.
The term humanized camelid antibody in the context of the present
specification refers to an
antibody consisting of only the heavy chain or the variable domain of the
heavy chain (VHH
domain) and whose amino acid sequence has been modified to increase their
similarity to
antibodies naturally produced in humans and, thus show a reduced
immunogenicity when
administered to a human being. A general strategy to humanize camelid
antibodies is shown
in Vincke et al., 2009, J Biol Chem., 284(5), 3273-3284, and US 2011/165621.
In the context of the present specification, the term fragment crystallizable
(Fc) region is used
in its meaning known in the art of cell biology and immunology; it refers to a
fraction of an
antibody comprising two identical heavy chain fragments comprised of a CH2 and
a CH3
domain, covalently linked by disulfide bonds.
The term specific binding in the context of the present invention refers to a
property of
ligands that bind to their target with a certain affinity and target
specificity. The affinity of such
a ligand is indicated by the dissociation constant of the ligand. A
specifically reactive ligand
has a dissociation constant of 5 10-7mo1/L when binding to its target, but a
dissociation
constant at least three orders of magnitude higher in its interaction with a
molecule having a

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globally similar chemical composition as the target, but a different three-
dimensional
structure.
In the context of the present specification, the term dissociation constant
(KD) is used in its
meaning known in the art of chemistry and physics; it refers to an equilibrium
constant that
measures the propensity of a complex composed of [mostly two] different
components to
dissociate reversibly into its constituent components. The complex can be e.g.
an antibody-
antigen complex AbAg composed of antibody Ab and antigen Ag. KD is expressed
in molar
concentration [mo1/1] and corresponds to the concentration of [Ab] at which
half of the binding
sites of [Ag] are occupied, in other words, the concentration of unbound [Ab]
equals the
concentration of the [AbAg] complex. The dissociation constant can be
calculated according
to the following formula:
[Ab] * [Ag]
KD = __________________________________________
[AbAg]
[Ab]: concentration of antibody; [Ag]: concentration of antigen; [AbAg]:
concentration of
antibodyantigen complex
In the context of the present specification, the terms off-rate (Koff11/secp
and on-rate (Kon;
[1/sec*M]) are used in their meaning known in the art of chemistry and
physics; they refer to
a rate constant that measures the dissociation (Koff) or association (Kon) of
5 an antibody
with its target antigen. Koff and Kon can be experimentally determined using
methods well
established in the art. A method for determining the Koff and Kon of an
antibody employs
surface plasmon resonance. This is the principle behind biosensor systems such
as the
Biacore or the ProteOn system. They can also be used to determine the
dissociation
constant KD by using the following formula:
[Koff 1
KD =
Won]
As used herein, the term pharmaceutical composition refers to a compound of
the invention,
or a pharmaceutically acceptable salt thereof, together with at least one
pharmaceutically
acceptable carrier. In certain embodiments, the pharmaceutical composition
according to the
invention is provided in a form suitable for topical, parenteral or injectable
administration.
As used herein, the term pharmaceutically acceptable carrier includes any
solvents,
dispersion media, coatings, surfactants, antioxidants, preservatives (for
example,
antibacterial agents, antifungal agents), isotonic agents, absorption delaying
agents, salts,
preservatives, drugs, drug stabilizers, binders, excipients, disintegration
agents, lubricants,
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sweetening agents, flavoring agents, dyes, and the like and combinations
thereof, as would
be known to those skilled in the art (see, for example, Remington: the Science
and Practice
of Pharmacy, ISBN 0857110624).
As used herein, the term treating or treatment of any disease or disorder
(e.g. cancer) refers
in one embodiment, to ameliorating the disease or disorder (e.g. slowing or
arresting or
reducing the development of the disease or at least one of the clinical
symptoms thereof). In
another embodiment "treating" or "treatment" refers to alleviating or
ameliorating at least one
physical parameter including those which may not be discernible by the
patient. In yet
another embodiment, "treating" or "treatment" refers to modulating the disease
or disorder,
either physically, (e.g., stabilization of a discernible symptom),
physiologically, (e.g.,
stabilization of a physical parameter), or both. Methods for assessing
treatment and/or
prevention of disease are generally known in the art, unless specifically
described
hereinbelow.
In the context of the present specification, the term dimer refers to a unit
consisting of two
subunits.
In the context of the present specification, the term homodimer refers to a
dimer comprised
of two subunits that are either identical or are highly similar members of the
same class of
subunits.
In the context of the present specification, the term amino acid linker refers
to a polypeptide
of variable length that is used to connect two polypeptides in order to
generate a single chain
polypeptide. Exemplary embodiments of linkers useful for practicing the
invention specified
herein are oligopeptide chains consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or
50 amino acids. A
non-limiting example of an amino acid linker is the polypeptide GGGGSGGGGS
(SEQ ID NO
83).
A first aspect of the invention relates to a tetrameric polypeptide
According to a first alternative of the first aspect, the tetrameric
polypeptide comprises or
consists of
- a first polypeptide chain comprising in N to C orientation a first VL
antigen binding
domain and a first CL constant domain,
- a second polypeptide chain comprising in N to C orientation a first VH
antigen binding
domain, a first CH1 constant domain, a first CH2 constant domain and a first
CH3
constant domain,
- a first ligand that specifically binds to a HER2 D4 epitope, wherein the
first ligand is
comprised in the first polypeptide chain and linked to the N-terminus of the
first VL
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antigen binding domain by a first interdomain amino acid linker, or the first
ligand is
comprised in the second polypeptide chain and linked to the N-terminus of the
first
VH antigen binding domain by a first interdomain amino acid linker,
- wherein the first VL antigen binding domain of the first polypeptide
chain and the first
VH antigen binding domain of the second polypeptide chain together constitute
a
second ligand, particularly a Fab domain, that specifically binds to a HER2 D1
epitope,
- a third polypeptide chain comprising in N to C orientation a second VL
antigen
binding domain and a second CL constant domain,
- a fourth polypeptide chain comprising in N to C orientation a second VH
antigen
binding domain, a second CH1 constant domain, a second CH2 constant domain and
a second CH3 constant domain,
- a third ligand that specifically binds to a HER2 D4 epitope, wherein the
third ligand is
comprised in the third polypeptide chain and linked to the N-terminus of the
second
VL antigen binding domain by a second interdomain amino acid linker, or the
third
ligand is comprised in the fourth polypeptide chain and linked to the N-
terminus of the
second VH antigen binding domain by a second interdomain amino acid linker,
- wherein the second VL antigen binding domain of the third polypeptide
chain and the
second VH antigen binding domain of the fourth polypeptide chain together
constitute
a fourth ligand, particularly an Fab domain, that specifically binds to a HER2
D1
epitope.
According to a second alternative of the first aspect, the tetrameric
polypeptide comprises or
consists of
- a first polypeptide chain comprising in N to C orientation a first VL
antigen binding
domain and a first CL constant domain,
- a second polypeptide chain comprising in N to C orientation a first VH
antigen binding
domain and a first CH1 constant domain, wherein particularly the second
polypeptide
chain further comprises a first CH2 constant domain, wherein more particularly
the
first CH2 constant domain is truncated at its C-terminus,
- a first ligand that specifically binds to a HER2 D4 epitope, wherein the
first ligand is
comprised in the first polypeptide chain and linked to the N-terminus of the
first VL
antigen binding domain by a first interdomain amino acid linker, or the first
ligand is
comprised in the second polypeptide chain and linked to the N-terminus of the
first
VH antigen binding domain by a first interdomain amino acid linker,
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- wherein the first VL antigen binding domain of the first polypeptide
chain and the first
VH antigen binding domain of the second polypeptide chain together constitute
a
second ligand, particularly a Fab domain, that specifically binds to a HER2 D1
epitope,
- a third polypeptide chain comprising in N to C orientation a second VL
antigen
binding domain and a second CL constant domain,
- a fourth polypeptide chain comprising in N to C orientation a second VH
antigen
binding domain and a second CH1 constant domain, wherein particularly the
fourth
polypeptide chain further comprises a second CH2 constant domain, wherein more
particularly the second CH2 constant domain is truncated at its C-terminus,
- a third ligand that specifically binds to a HER2 D4 epitope, wherein the
third ligand is
comprised in the third polypeptide chain and linked to the N-terminus of the
second
VL antigen binding domain by a second interdomain amino acid linker, or the
third
ligand is comprised in the fourth polypeptide chain and linked to the N-
terminus of the
second VH antigen binding domain by a second interdomain amino acid linker,
- wherein the second VL antigen binding domain of the third polypeptide
chain and the
second VH antigen binding domain of the fourth polypeptide chain together
constitute
a fourth ligand, particularly an Fab domain, that specifically binds to a HER2
D1
epitope.
All embodiments below may be combined with both the first alternative and the
second
alternative of the first aspect of the invention.
The VL antigen binding domain and the CL constant domain of the first and
third polypeptide
chain are domains of an immunoglobulin G light chain, and the VH antigen
binding domain
and the CH1, CH2 and CH3 constant domains of the second and fourth polypeptide
are
domains of an immunoglobulin G heavy chain. In other words, the first and the
third
polypeptide chains each comprise an immunoglobulin G light chain, and the
second and the
fourth polypeptide chains each comprise an immunoglobulin G heavy chain. The
immunoglobulin light and heavy chains of the tetrameric polypeptide according
to the
invention form the second and fourth ligands specifically binding to HER2 D1
epitope.
In addition, polypeptides comprising a first and third ligand which
specifically binds to HER2
D4 epitope are fused to the N-terminus of the immunoglobulin G heavy chains or
immunoglobulin G light chains by an interdomain amino acid linker resulting in
a tetrameric
polypeptide having four HER2 binding sites, two of which bind to the D4
epitope and two of
which bind to the D1 epitope.
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Surprisingly, the tetrameric polypeptide according to the invention displays
superior HER2
inactivation compared to conventional antibodies and other antibody-like
molecules, such as
divalent biparatopic polypeptides (having a total of two binding regions) and
has additional
effects on cell growth, apoptosis, HER2 internalization and HER2 degradation.
Therefore, the
tetrameric polypeptides according to the present invention are promising
candidates for
therapy of HER2-expressing cancer.
Without wishing to be bound be theory, it is believed that the CH1, CH2, CH3
and CL
domains contribute, particularly the CH2 and CH3 domains, to the additional
effects of the
tetrameric polypeptides of the invention, particularly inhibition of cell
growth and proliferation,
induction of apoptosis and other forms of cell death, HER2 internalization,
HER2 recycling
inhibition and HER2 degradation, HER2 crosslinking, reduction of HER2 surface
mobility,
HER2 expression downregulation, inhibition of HER2 dimerization and signalling
by
positioning the variable domains at a particular angle and distance.
In certain embodiments, the first polypeptide chain and the third polypeptide
chain comprise
a sequence identity with each other of 70% or more, particularly 80% or more,
more
particularly 90% or more, even more particularly 95% or more, wherein most
particularly the
first polypeptide chain and the third polypeptide chain are identical.
In certain embodiments, the first polypeptide chain comprises a sequence
identity of 70%,
71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
88%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with
the
third polypeptide chain.
In certain embodiments, the second polypeptide chain and the fourth
polypeptide chain
comprise a sequence identity with each other of 70% or more, particularly 80%
or more,
more particularly 90% or more, even more particularly 95% or more, wherein
most
particularly the second polypeptide chain and the fourth polypeptide chain are
identical.
In certain embodiments, the second polypeptide chain comprises a sequence
identity of
70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
with the fourth polypeptide chain.
In certain embodiments, the immunoglobulin light chain of the first
polypeptide chain and the
immunoglobulin light chain of the third polypeptide chain comprise a sequence
identity with
each other of 70% or more, particularly 80% or more, more particularly 90% or
more, even
more particularly 95% or more, wherein most particularly the immunoglobulin
light chain of
the first polypeptide chain and the immunoglobulin light chain of the third
polypeptide chain
are identical.

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In certain embodiments, the immunoglobulin light chain of the first
polypeptide chain
comprises a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99% or 100% with the immunoglobulin light chain of the third
polypeptide
chain.
In certain embodiments, the immunoglobulin heavy chain of the second
polypeptide chain
and the immunoglobulin heavy chain of the fourth polypeptide chain comprise a
sequence
identity with each other of 70% or more, particularly 80% or more, more
particularly 90% or
more, even more particularly 95% or more, wherein most particularly the
immunoglobulin
heavy chain of the second polypeptide chain and the immunoglobulin heavy chain
of the
fourth polypeptide chain are identical.
In certain embodiments, the immunoglobulin heavy chain of the second
polypeptide chain
comprises a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, 99% or 100% with the immunoglobulin heavy chain of the fourth
polypeptide chain.
In certain embodiments, the first ligand and the third ligand comprise a
sequence identity
with each other of 70% or more, particularly 80% or more, more particularly
90% or more,
even more particularly 95% or more, wherein most particularly the first ligand
and the third
ligand are identical.
In certain embodiments, the first ligand comprises a sequence identity of 70%,
71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the third
ligand.
In other words, the first ligand is substantially the same as the third
ligand.
In certain embodiments, the second ligand and the fourth ligand comprise a
sequence
identity with each other of 70% or more, particularly 80% or more, more
particularly 90% or
more, even more particularly 95% or more, wherein most particularly the first
ligand and the
third ligand are identical.
In certain embodiments, the second ligand comprises a sequence identity of
70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the
fourth
ligand.
In other words, the second ligand is substantially the same as the fourth
ligand.
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In certain embodiments, the first CH2 constant domain and the first CH3
constant domain of
the second polypeptide chain interact with the second CH2 constant domain and
the second
CH3 constant domain of the fourth polypeptide chain, such that a tetrameric
polypeptide is
formed. In particular, the CH2 and CH3 constant domains of the second and
fourth
polypeptide chain dimerize. In particular, combined with the interaction
between the first and
the second polypeptide chains and the third and the fourth polypeptide chains,
particularly by
means of the dimerization between the respective VL and VH antigen binding
domains and
the CL and CH1 constant domains, this results in tetramer formation of the
first, second, third
and fourth polypeptide. Therefore, in respect of multimer formation, the
tetrameric
polypeptide of the present invention is similar to an antibody.
In certain embodiments, the second polypeptide chain comprises a first hinge
region
between the first CH1 constant domain and the first CH2 constant domain, and
the fourth
polypeptide chain comprises a second hinge region between the second CH1
constant
domain and the second CH2 constant domain, wherein the first hinge region and
the second
hinge region mediate complex formation between the second polypeptide chain
and the
fourth polypeptide chain, particularly by at least one disulphide bond, more
particularly by a
first disulphide bond and a second disulphide bond, such that a tetrameric
polypeptide is
formed. Complex formation between the CH1 and CH2 constant domains may thus
occur by
the hinge region, i.e. by disulphide bond formation between cysteine residues,
just as in
antibodies.
In certain embodiments, the CH2 constant domain and/or the CH3 constant domain
of the
second polypeptide chain is truncated at its C-terminus.
In certain embodiments, the CH2 constant domain and/or the CH3 constant domain
of the
fourth polypeptide chain is truncated at its C-terminus.
In certain embodiments, the first ligand comprises or consists of a single-
chain variable
fragment polypeptide chain comprising an scFy heavy chain, an scFy linker
chain, and an
scFy light chain. In certain embodiments, the scFy heavy chain is the VH
domain of 4D5
(particularly SEQ ID No. 80), and wherein the scFy light chain is the VL
domain of 4D5
(particularly SEQ ID No. 81).
In certain embodiments, the single-chain variable fragment polypeptide chain
comprises in N
to C orientation an scFy heavy chain, an scFy linker chain, and an scFy light
chain.
In certain embodiments, the single-chain variable fragment polypeptide chain
comprises in N
to C orientation an scFy light chain, an scFy linker chain, and an scFy heavy
chain.
In other words, the the scFy heavy chain and the scFy light chain may be
provided in any
orientation on the single-chain variable fragment polypeptide chain.
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In certain embodiments, the third ligand comprises or consists of a single-
chain variable
fragment polypeptide chain comprising in N to C orientation an scFv heavy
chain, an scFv
linker chain, and an scFv light chain. In certain embodiments, the scFv heavy
chain is the VH
domain of 4D5 (particularly SEQ ID No. 80), and wherein the scFv light chain
is the VL
domain of 4D5 (particularly SEQ ID No. 81).
In certain embodiments,
- the scFv heavy chain of the first ligand comprises or consists of a
peptide sequence
having a sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or more, even more particularly 95 % or more, with a peptide
sequence selected from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No.
23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, wherein
most particularly the scFv heavy chain of the first ligand comprises or
consists of a
peptide sequence identical to a peptide sequence selected from SEQ ID No. 15,
SEQ
ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID
No. 54 and SEQ ID No. 80, and
- the scFv light chain of the first ligand comprises or consists of a
peptide sequence
having a sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or more, even more particularly 95 % or more, with a peptide
sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No.
26, SEQ ID No. 43 and SEQ ID No. 81, wherein most particularly the scFv light
chain
of the first ligand comprises or consists of a peptide sequence identical to a
peptide
sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No.
26, SEQ ID No. 43 and SEQ ID No. 81.
In certain embodiments,
- the scFv heavy chain of the first ligand comprises or consists of a peptide
sequence
having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected
from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No.
44,
SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, and
- the scFv light chain of the first ligand comprises or consists of a
peptide sequence
having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected
from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43
and SEQ ID No. 81.
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In certain embodiments,
- the scFv heavy chain of the third ligand comprises or consists of a
peptide sequence
having a sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or more, even more particularly 95 % or more, with a peptide
sequence selected from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No.
23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, wherein
most particularly the scFv heavy chain of the third ligand comprises or
consists of a
peptide sequence identical to a peptide sequence selected from SEQ ID No. 15,
SEQ
ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID
No. 54 and SEQ ID No. 80, and
- the scFv light chain of the third ligand comprises or consists of a
peptide sequence
having a sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or more, even more particularly 95 % or more, with a peptide
sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No.
26, SEQ ID No. 43 and SEQ ID No. 81, wherein most particularly the scFv light
chain
of the third ligand comprises or consists of a peptide sequence identical to a
peptide
sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No.
26, SEQ ID No. 43 and SEQ ID No. 81.
In certain embodiments,
- the scFv heavy chain of the third ligand comprises or consists of a peptide
sequence
having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected
from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No.
44,
SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, and
- the scFv light chain of the third ligand comprises or consists of a
peptide sequence
having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected
from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43
and SEQ ID No. 81.
In certain embodiments,
- the scFv heavy chain of the first ligand and the third ligand each
comprises or
consists of a peptide sequence having a sequence identity of 70 % or more,
particularly 80 % or more, more particularly 90 % or more, even more
particularly 95
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% or more, with a peptide sequence selected from SEQ ID No. 15, SEQ ID No. 21,
SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and
SEQ ID No. 80, wherein most particularly the scFy heavy chain of the first
ligand and
the third ligand each comprises or consists of a peptide sequence identical to
a
peptide sequence selected from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22,
SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80,
and
- the scFy light chain of the first ligand and the third ligand each
comprises or consists
of a peptide sequence having a sequence identity of 70 % or more, particularly
80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with a
peptide sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25,
SEQ ID No. 26, SEQ ID No. 43 and SEQ ID No. 81, wherein most particularly the
scFy light chain of the first ligand and the third ligand each comprises or
consists of a
peptide sequence identical to a peptide sequence selected from SEQ ID No. 14,
SEQ
ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43 and SEQ ID No. 81.
In certain embodiments,
- the scFy heavy chain of the first ligand and the third ligand each
comprises or
consists of a peptide sequence having a sequence identity of 70%, 71%, 72%,
73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a
peptide sequence selected from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22,
SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80,
and
- the scFy light chain of the first ligand and the third ligand each
comprises or consists
of a peptide sequence having a sequence identity of 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a
peptide sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25,
SEQ ID No. 26, SEQ ID No. 43 and SEQ ID No. 81.
That is the scFy light and heavy chains may completely consist of the above-
defined peptide
sequence or the scFv light and heavy chains may comprise the above-defined
peptide
sequence, wherein the scFy light and heavy chains may contain additional
peptide
sequences.
In certain embodiments, the scFy light chain of the first ligand comprises or
consists of the
VL antigen binding domain of antibody 4D5 and the scFy heavy chain of the
first ligand
comprises or consists of the VH antigen binding domain of antibody 4D5.

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In certain embodiments, the scFv light chain of the third ligand comprises or
consists of the
VL antigen binding domain of antibody 4D5 and the scFv heavy chain of the
third ligand
comprises or consists of the VH antigen binding domain of antibody 4D5.
In the context of the present specification, the term 4D5 refers to the
humanized monoclonal
antibody trastuzumab, also known as Herceptin, and also referred to herein as
"TZB" which
is directed against the membrane-proximal domain IV of HER2 (Cho et al.,
2003).
In certain embodiments, the scFv linker chain comprises a peptide sequence
having a
sequence identity of 70 % or more, particularly 80 % or more, more
particularly 90 % or
more, even more particularly 95 % or more, with SEQ ID No. 16, wherein most
particularly
the scFv linker chain comprises a peptide sequence identical to SEQ ID No. 16.
In certain embodiments, the scFv linker chain comprises a peptide sequence
having a
sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%,
98%, 99% or 100% with SEQ ID No. 16.
In certain embodiments,
- the first polypeptide chain comprises a peptide sequence having a
sequence identity
of 70 % or more, particularly 80 % or more, more particularly 90 % or more,
even
more particularly 95 % or more, with a peptide sequence selected from SEQ ID
No.
18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41,
SEQ ID No. 50 and SEQ ID 76, wherein most particularly the first polypeptide
chain
comprises a peptide sequence identical to a peptide sequence selected from SEQ
ID
No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No.
41, SEQ ID No. 50 and SEQ ID 76, and
- the second polypeptide chain comprises a peptide sequence having a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with a peptide sequence selected from SEQ
ID
No. 19, SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No.
40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77, wherein most
particularly the second polypeptide chain comprises a peptide sequence
identical to a
peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27,
SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51,
SEQ ID No. 52 and SEQ ID 77.
In certain embodiments,
- the first polypeptide chain comprises a peptide sequence having a
sequence identity
of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
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84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% with a peptide sequence selected from SEQ ID No. 18, SEQ ID
No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No.
50 and SEQ ID 76,
- the second polypeptide chain comprises a peptide sequence having a sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID No. 19,
SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40,
SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77.
In certain embodiments,
- the third polypeptide chain comprises a peptide sequence having a sequence
identity
of 70 % or more, particularly 80 % or more, more particularly 90 % or more,
even
more particularly 95 % or more, with a peptide sequence selected from SEQ ID
No.
18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41,
SEQ ID No. 50 and SEQ ID 76, wherein most particularly the third polypeptide
chain
comprises a peptide sequence identical to a peptide sequence selected from SEQ
ID
No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No.
41, SEQ ID No. 50 and SEQ ID 76, and
- the fourth polypeptide chain comprises a peptide sequence having a sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with a peptide sequence selected from SEQ
ID
No. 19, SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No.
40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77, wherein most
particularly the fourth polypeptide chain comprises a peptide sequence
identical to a
peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27,
SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51,
SEQ ID No. 52 and SEQ ID 77.
In certain embodiments,
- the third polypeptide chain comprises a peptide sequence having a sequence
identity
of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% with a peptide sequence selected from SEQ ID No. 18, SEQ ID
No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No.
50 and SEQ ID 76,
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- the fourth polypeptide chain comprises a peptide sequence having a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID No. 19,
SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40,
SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each
comprises a peptide
sequence having a sequence identity of 70 % or more, particularly 80 % or
more,
more particularly 90 % or more, even more particularly 95 % or more, with a
peptide
sequence selected from SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No.
32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 50 and SEQ ID 76, wherein most
particularly the first polypeptide chain and the third polypeptide chain each
comprises
a peptide sequence identical to a peptide sequence selected from SEQ ID No.
18,
SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41,
SEQ ID No. 50 and SEQ ID 76, and
- the second polypeptide chain and the fourth polypeptide chain each
comprises a
peptide sequence having a sequence identity of 70 % or more, particularly 80 %
or
more, more particularly 90 % or more, even more particularly 95 % or more,
with a
peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27,
SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51,
SEQ ID No. 52 and SEQ ID 77, wherein most particularly the second polypeptide
chain and the fourth polypeptide chain each comprises a peptide sequence
identical
to a peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No.
27,
SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51,
SEQ ID No. 52 and SEQ ID 77.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each
comprises a peptide
sequence having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence
selected from SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ
ID No. 39, SEQ ID No. 41, SEQ ID No. 50 and SEQ ID 76,
- the second polypeptide and the fourth polypeptide chain each comprises a
peptide
sequence having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%,
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77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence
selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ
ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ
1D77.
Therefore, the VL and VH antigen binding domain of the first, second, third
and fourth
polypeptide chains may be substantially identical to the VL and VH antigen
binding domains
of the scFv fragment termed A21 (Hu S. et al., 2008, Proteins, 70, 938-949)
which
specifically binds to domain I of HER2.
In certain embodiments,
- the first polypeptide chain comprises a peptide sequence having a
sequence identity
of 70 % or more, particularly 80 % or more, more particularly 90 % or more,
even
more particularly 95 % or more, with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No.
38
and SEQ ID No. 78, wherein most particularly the first polypeptide chain
comprises a
peptide sequence identical to SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and
SEQ ID No. 78, and
- the second polypeptide chain comprises a peptide sequence having a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 35 and SEQ ID No. 79, wherein most particularly the second polypeptide
chain
comprises a peptide sequence identical to SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 35 and SEQ ID No. 79.
In certain embodiments,
- the first polypeptide chain comprises a peptide sequence having a
sequence identity
of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID
No. 78, and
- the second polypeptide chain comprises a peptide sequence having a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and
SEQ ID No. 79.
In certain embodiments,
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- the third polypeptide chain comprises a peptide sequence having a
sequence identity
of 70 % or more, particularly 80 % or more, more particularly 90 % or more,
even
more particularly 95 % or more, with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No.
38
and SEQ ID No. 78, wherein most particularly the third polypeptide chain
comprises a
peptide sequence identical to SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and
SEQ ID No. 78, and
- the fourth polypeptide chain comprises a peptide sequence having a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 35 and SEQ ID No. 79, wherein most particularly the fourth polypeptide
chain
comprises a peptide sequence identical to SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 35 and SEQ ID No. 79.
In certain embodiments,
- the third polypeptide chain comprises a peptide sequence having a
sequence identity
of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID
No. 78, and
- the fourth polypeptide chain comprises a peptide sequence having a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and
SEQ ID No. 79.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each comprises a
peptide
sequence having a sequence identity of 70 % or more, particularly 80 % or
more,
more particularly 90 % or more, even more particularly 95 % or more, with SEQ
ID
No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, wherein most
particularly
the first polypeptide chain and the third polypeptide chain each comprises a
peptide
sequence identical to SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID
No. 78, and
- the second polypeptide chain and the fourth polypeptide chain each
comprises a
peptide sequence having a sequence identity of 70 % or more, particularly 80 %
or
more, more particularly 90 % or more, even more particularly 95 % or more,
with SEQ
ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79, wherein most

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particularly the second polypeptide chain and the fourth polypeptide chain
each
comprises a peptide sequence identical to SEQ ID No. 33, SEQ ID No. 34, SEQ ID
No. 35 and SEQ ID No. 79.
In certain embodiments,
- the
first polypeptide chain and the third polypeptide chain each comprises a
peptide
sequence having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99% or 100% with SEQ ID No. 36,
SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, and
- the second polypeptide chain and the fourth polypeptide chain each comprises
a
peptide sequence having a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%,
78%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99% or 100% with SEQ ID No.
33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79.
Therefore, the immunoglobulin light and heavy chains of the first, second,
third and fourth
polypeptide chains may comprise IgG domains (VL, CL, VH, CH1, CH2 and/or CH3)
substantially identical to the corresponding domains of the ErbB2 antibody
termed 702 (US
7,371,376) which specifically binds to domain I of HER2.
In certain particular embodiments, the tetrameric polypeptide according to the
invention is
essentially an immunoglobulin G type antibody (particularly a human or
humanized
monoclonal IgG antibody) having two identical heavy chains and two identical
light chains,
wherein the antigen specific variable heavy and light chains together form a
ligand (the
second and fourth ligand) specifically reactive to the D1 domain of Her2, and
each of the light
chains, or each of the heavy chains, contain an N-terminally linked
polypeptide comprising
an scFv polypeptide chain constituted of a heavy and light variable region
linked by an scFy
linker chain, and the scFy polypeptide chain is linked to the N terminus of
the
immunoglobulin heavy or light chain via an interdomain amino acid linker
consisting of 1 to
20 amino acids.
In certain embodiments,
- the first polypeptide chain has a sequence identity of 70 % or more,
particularly 80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with
SEQ ID No. 1, wherein most particularly the first polypeptide chain is
identical to SEQ
ID No. 1, and
-
the second polypeptide chain has a sequence identity of 70 % or more,
particularly 80
% or more, more particularly 90 % or more, even more particularly 95 % or
more, with
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SEQ ID No. 2, wherein most particularly the second polypeptide chain is
identical to
SEQ ID No. 2.
In certain embodiments,
- the first polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 1, and
- the second polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 2.
In certain embodiments,
- the third polypeptide chain has a sequence identity of 70 % or more,
particularly 80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with
SEQ ID No. 1, wherein most particularly the third polypeptide chain is
identical to
SEQ ID No. 1, and
- the fourth polypeptide chain has a sequence identity of 70 % or more,
particularly 80
% or more, more particularly 90 % or more, even more particularly 95 % or
more, with
SEQ ID No. 2, wherein most particularly the fourth polypeptide chain is
identical to
SEQ ID No. 2.
In certain embodiments,
- the third polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 1, and
- the fourth polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 2.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each have a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 1, wherein most
particularly
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the first polypeptide chain and the third polypeptide chain are identical to
SEQ ID No.
1, and
- the second polypeptide chain and the fourth polypeptide chain each have a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 2, wherein most
particularly
the second polypeptide chain and the fourth polypeptide chain are identical to
SEQ ID
No. 2.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each have a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%,
97%, 98%, 99% or 100% with SEQ ID No. 1, and
- the second polypeptide chain and the fourth polypeptide chain each have a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%,
97%, 98%, 99% or 100% with SEQ ID No. 2.
The resulting tetrameric polypeptide is referred to as "441" (for identity).
In this construct,
IgG light chains (of the first and third polypeptide chain) comprising the VL
antigen
binding domain of antibody A21 are N-terminally fused to an scFv fragment
comprising
the VL and VH antigen binding domains of 4D5 (trastuzumab or HERCEPTIN, HER2
D4
binder) and combined with IgG heavy chains (the second and fourth polypeptide
chains)
comprising the VH antigen binding domain of antibody A21. The VL and VH
antigen
binding domains of A21 together constitute a HER2 D1 binder).
In certain embodiments,
- the first polypeptide chain has a sequence identity of 70 % or more,
particularly 80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with
SEQ ID No. 3, wherein most particularly the first polypeptide chain is
identical to SEQ
ID No. 3, and
- the second polypeptide chain has a sequence identity of 70 % or more,
particularly 80
% or more, more particularly 90 % or more, even more particularly 95 % or
more, with
SEQ ID No. 4, wherein most particularly the second polypeptide chain is
identical to
SEQ ID No. 4.
In certain embodiments,
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- the first polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 3, and
- the
second polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 4.
In certain embodiments,
- the third polypeptide chain has a sequence identity of 70 % or more,
particularly 80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with
SEQ ID No. 3, wherein most particularly the third polypeptide chain is
identical to
SEQ ID No. 3, and
- the fourth polypeptide chain has a sequence identity of 70 % or more,
particularly 80
% or more, more particularly 90 % or more, even more particularly 95 % or
more, with
SEQ ID No. 4, wherein most particularly the fourth polypeptide chain is
identical to
SEQ ID No. 4.
In certain embodiments,
- the third polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 3, and
- the fourth polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 4.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each have a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 3, wherein most
particularly
the first polypeptide chain and the third polypeptide chain are identical to
SEQ ID No.
3, and
- the second polypeptide chain and the fourth polypeptide chain each have a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
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even more particularly 95 % or more, with SEQ ID No. 4, wherein most
particularly
the second polypeptide chain and the fourth polypeptide chain are identical to
SEQ ID
No. 4.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each have a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%,
97%, 98%, 99% or 100% with SEQ ID No. 3, and
- the second polypeptide chain and the fourth polypeptide chain each
have a sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%,
97%, 98%, 99% or 100% with SEQ ID No. 4.
The resulting tetrameric polypeptide is referred to as "4702" (for identity).
In this
construct, IgG light chains (of the first and third polypeptide chain)
comprising the VL
antigen binding domain of antibody 702 are N-terminally fused to an scFv
fragment
comprising the VL and VH antigen binding domains of 4D5 (trastuzumab or
HERCEPTIN,
HER2 D4 binder) and combined with IgG heavy chains (the second and fourth
polypeptide chains) comprising the VH antigen binding domain of antibody 702.
The VL
and VH antigen binding domains of 702 together constitute a HER2 D1 binder).
In certain embodiments,
- the first polypeptide chain has a sequence identity of 70 % or more,
particularly 80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with
SEQ ID No. 11, wherein most particularly the first polypeptide chain is
identical to
SEQ ID No. 11, and
- the second polypeptide chain has a sequence identity of 70 % or more,
particularly 80
% or more, more particularly 90 % or more, even more particularly 95 % or
more, with
SEQ ID No. 12, wherein most particularly the second polypeptide chain is
identical to
SEQ ID No. 12.
In certain embodiments,
- the first polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%,
74%,
75%, 78%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99% or 100% with SEQ ID
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- the second polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 12.
In certain embodiments,
- the third polypeptide chain has a sequence identity of 70 % or more,
particularly 80 %
or more, more particularly 90 % or more, even more particularly 95 % or more,
with
SEQ ID No. 11, wherein most particularly the third polypeptide chain is
identical to
SEQ ID No. 11, and
- the fourth polypeptide chain has a sequence identity of 70 % or more,
particularly 80
% or more, more particularly 90 % or more, even more particularly 95 % or
more, with
SEQ ID No. 12, wherein most particularly the fourth polypeptide chain is
identical to
SEQ ID No. 12.
In certain embodiments,
- the third polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%,
74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 11, and
- the fourth polypeptide chain has a sequence identity of 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID
No. 12.
In certain embodiments,
- the first polypeptide chain and the third polypeptide chain each have a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 11, wherein most
particularly
the first polypeptide chain and the third polypeptide chain are identical to
SEQ ID No.
11, and
- the second polypeptide chain and the fourth polypeptide chain each have a
sequence
identity of 70 % or more, particularly 80 % or more, more particularly 90 % or
more,
even more particularly 95 % or more, with SEQ ID No. 12, wherein most
particularly
the second polypeptide chain and the fourth polypeptide chain are identical to
SEQ ID
No. 12.
In certain embodiments,
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- the first polypeptide chain and the third polypeptide chain each have a
sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% with SEQ ID No. 11, and
- the
second polypeptide chain and the fourth polypeptide chain each have a sequence
identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% with SEQ ID No. 12.
The resulting tetrameric polypeptide is referred to as "241" (for identity).
In this construct,
IgG heavy chains (of the second and fourth polypeptide chain) comprising the
VH antigen
binding domain of antibody A21 are N-terminally fused to an scFv fragment
comprising
the VL and VH antigen binding domains of 4D5 (trastuzumab or HERCEPTIN, HER2
D4
binder) and combined with IgG light chains (the first and third polypeptide
chains)
comprising the VL antigen binding domain of antibody A21. The VL and VH
antigen
binding domains of A21 together constitute a HER2 D1 binder).
In certain embodiments, the VH antigen binding domain is selected from the VH
antigen
binding domain of A21 (particularly SEQ ID No. 40, 42, 51, 52 or 77) and the
VH antigen
binding domain of 702 (particularly SEQ ID No. 79), and wherein the VL antigen
binding
domain is selected from the VL antigen binding domain of A21 (particularly SEQ
ID No. 39,
41, 50 or 76) and the VL antigen binding domain of 702 (particularly SEQ ID
No. 78).
In certain embodiments, the first and third polypeptide chains are identical
to SEQ ID No. 1
or a functional equivalent peptide sequence having a sequence identity of at
least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and the second and fourth
polypeptide
chains are identical to SEQ ID No. 2 or a functional equivalent peptide
sequence having a
sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99%
(construct 441 in case of sequence identity).
In certain embodiments, the first and third polypeptide chains are identical
to SEQ ID No. 3
or a functional equivalent peptide sequence having a sequence identity of at
least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and the second and fourth
polypeptide
chains are identical to SEQ ID No. 4 or a functional equivalent peptide
sequence having a
sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99%
(construct 4702 in case of sequence identity).
In certain embodiments, the first and third polypeptide chains are identical
to SEQ ID No. 11
or a functional equivalent peptide sequence having a sequence identity of at
least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and the second and fourth
polypeptide
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chains are identical to SEQ ID No. 12 or a functional equivalent peptide
sequence having a
sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99%
(construct 241 in case of sequence identity).
The interdomain amino acid linker is not restricted in amino acid composition
but amino acids
shown to contribute to linker flexibility are chosen in particular embodiments
contemplated
herein. The inventors have shown linkers to work that consist of G, S and/or T
residues, for
example repeats of (GG,,S) and (GG,,T) with m selected from 1 to 3, and the
entire linker
length not exceeding 20, 25, or even 30. lnterdomain linkers as short as one
or two amino
acids have been shown to work. The first and the third polypeptide may
comprise the same
interdomain amino acid linker or different interdomain amino acid linkers.
In certain embodiments, the interdomain amino acid linker consists of 1, 2, 3,
4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.
With regard to the length and sequence composition of the interdomain amino
acid linker, the
inventors' results indicate that any linker having an equivalent length of a
maximum of 25
.. amino acids that is not expected, because of structure prediction, to
interfere with the
solubility of the resulting protein, is expected to function.
The polypeptide according to any one of the preceding claims, wherein the
interdomain
amino acid linker comprises or consists of amino acids G, A, J, S, T, P, C, V,
M and E,
particularly wherein the interdomain amino acid linker comprises or consists
of amino acids
G, S, A and T.
In particular embodiments, the interdomain amino acid linker is (GGE)n with n
being an
integer and n 4 (particularly n is 4, 5, 6, 7 or 8), and with E selected from
S and T.
In particular embodiments, the interdomain amino acid linker is (GGS)n with n
being an
integer and n 4 (particularly n is 4, 5, 6, 7 or 8).
In particular embodiments, the interdomain amino acid linker is (GGT)n with n
being an
integer and n 4 (particularly n is 4, 5, 6, 7 or 8).
In particular embodiments, the interdomain amino acid linker is (GEG)n with n
being an
integer and n 4 (particularly n is 4, 5, 6, 7 or 8), and with E selected from
S and T.
In particular embodiments, the interdomain amino acid linker is (FFE)n with n
being an integer
and n 4 (particularly n is 4, 5, 6, 7 or 8), and with each F independently
from any other F
being selected being from A, G and V, and E being selected from S and T.
Important considerations at the time of choosing the linker sequence have been
solubility
and flexibility. The skilled person will readily be able to vary this sequence
in composition and
length based on the teaching herein and the knowledge available on linker
design, as
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exemplified by Chen et al., 2013, Advanced Drug Delivery Reviews, 65, 1357-
1369 and
Evers et al., 2006, Biochemistry, 45, 13183-13192.
In certain embodiments, the interdomain amino acid linker is characterized by
an amino acid
sequence (GGGGS)n, with n being 1, 2, 3, 4 or 5.
In certain embodiments, the interdomain amino acid linker comprises or is a
sequence
characterized by one of SEQ ID No. 17, SEQ ID No. 55 to SQ ID No. 69 and SEQ
ID No. 82
to SEQ ID 91.
In certain embodiments, the interdomain amino acid linker comprises or
consists of a peptide
sequence selected from one of SEQ ID No. 17, SEQ ID No. 55 to SEQ ID No. 69
and SEQ
ID No. 82 to SEQ ID No. 91 or a functional equivalent peptide sequence having
a sequence
identity of at least 70%.
A second aspect of the invention relates to the polypeptide according to the
first aspect for
use in a method for the prevention or treatment of a malignant neoplastic
disease associated
with expression of HER2 (a HER2-positive cancer).
Similarly, a dosage form for the prevention or treatment of a malignant
neoplastic disease
associated with expression of HER2 is provided, comprising a tetrameric
polypeptide of the
invention.
The skilled person is aware that any specifically mentioned drug may be
present as a
pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable
salts comprise
the ionized drug and an oppositely charged counterion. Non-limiting examples
of
pharmaceutically acceptable anionic salt forms include acetate, benzoate,
besylate, bitatrate,
bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate,
fumarate,
gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate,
lactobionate, malate,
maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,
napsylate, nitrate,
pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate,
succinate, sulfate,
tartrate, tosylate, triethiodide and valerate. Non-limiting examples of
pharmaceutically
acceptable cationic salt forms include aluminium, benzathine, calcium,
ethylene diamine,
lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and
zinc.
Dosage forms may be for enteral administration, such as nasal, buccal, rectal,
transdermal or
oral administration, or as an inhalation form or suppository. Alternatively,
parenteral
administration may be used, such as subcutaneous, intravenous, intrahepatic or
intramuscular injection forms. Optionally, a pharmaceutically acceptable
carrier and/or
excipient may be present.
Topical administration is also within the scope of the advantageous uses of
the invention.
The skilled artisan is aware of a broad range of possible recipes for
providing topical
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formulations, as exemplified by the content of Benson and Watkinson (Eds.),
Topical and
Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011,
ISBN-13: 978-
0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised
and
Expanded (2nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann
(Eds.): Topical Drug Delivery Formulations (1st Ed. CRC Press 1989; ISBN-13:
978-
0824781835).
A third aspect of the invention relates to an isolated nucleic acid encoding
at least one of the
first polypeptide chain, the second polypeptide chain, the third polypeptide
chain and the
fourth polypeptide chain of the tetrameric polypeptide according to the first
aspect of the
invention. In particular, the isolated nucleic acid may be comprised in a
plasmid for
expression in a bacterial or a eukaryotic host cell. The nucleic acid
sequences encoding the
first, the second, the third and the fourth polypeptide may be provided on the
same plasmid
or on separate plasmids, i. e for co-expression in the same host.
A fourth aspect of the invention relates to a host cell which is adapted to
produce at least one
of the first polypeptide chain, the second polypeptide chain, the third
polypeptide chain and
the fourth polypeptide chain of the tetrameric polypeptide according to the
first aspect of the
invention. In particular, the host cell is a bacterial cell or a eukaryotic
cell. More particularly,
the host cell is a Chinese Hamster Ovary (CHO) cell.
In certain embodiments, the host cell comprises the isolated nucleic acid
according to the
third aspect of the invention, such that the host cell is able to produce at
least one of the first
polypeptide chain, the second polypeptide chain, the third polypeptide chain
and the fourth
polypeptide chain of the polypeptide according to the first aspect of the
invention. In
particular, the first, second, third and fourth polypeptide may be co-produced
in the same cell
or produced separately and combined in vitro.
.. A fifth aspect of the invention relates to a method for obtaining the
polypeptide according to
the first aspect of the invention, wherein the method comprises culturing the
host cell
according to the fourth aspect of the invention, so that at least one of the
first polypeptide
chain, the second polypeptide chain, the third polypeptide chain and the
fourth polypeptide
chain of the polypeptide according to the first aspect of the invention is
produced.
Pharmaceutical Composition and Administration
Another aspect of the invention relates to a pharmaceutical composition
comprising a
tetrameric polypeptide of the present invention, or a pharmaceutically
acceptable salt thereof,
and a pharmaceutically acceptable carrier. In further embodiments, the
composition
comprises at least two pharmaceutically acceptable carriers, such as those
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In certain embodiments of the invention, the tetrameric polypeptide of the
present invention is
typically formulated into pharmaceutical dosage forms to provide an easily
controllable
dosage of the drug and to give the patient an elegant and easily handleable
product.
In embodiments of the invention relating to topical uses of the tetrameric
polypeptide of the
invention, the pharmaceutical composition is formulated in a way that is
suitable for topical
administration such as aqueous solutions, suspensions, ointments, creams, gels
or
sprayable formulations, e.g., for delivery by aerosol or the like, comprising
the active
ingredient together with one or more of solubilizers, stabilizers, tonicity
enhancing agents,
buffers and preservatives that are known to those skilled in the art.
The pharmaceutical composition can be formulated for oral administration,
parenteral
administration, or rectal administration. In addition, the pharmaceutical
compositions of the
present invention can be made up in a solid form (including without limitation
capsules,
tablets, pills, granules, powders or suppositories), or in a liquid form
(including without
limitation solutions, suspensions or emulsions).
The dosage regimen for the compounds of the present invention will vary
depending upon
known factors, such as the pharmacodynamic characteristics of the particular
agent and its
mode and route of administration; the species, age, sex, health, medical
condition, and
weight of the recipient; the nature and extent of the symptoms; the kind of
concurrent
treatment; the frequency of treatment; the route of administration, the renal
and hepatic
function of the patient, and the effect desired. In certain embodiments, the
compounds of the
invention may be administered in a single daily dose, or the total daily
dosage may be
administered in divided doses of two, three, or four times daily.
The pharmaceutical compositions of the present invention can be subjected to
conventional
pharmaceutical operations such as sterilization and/or can contain
conventional inert
diluents, lubricating agents, or buffering agents, as well as adjuvants, such
as preservatives,
stabilizers, wetting agents, emulsifiers and buffers, etc. They may be
produced by standard
processes, for instance by conventional mixing, granulating, dissolving or
lyophilizing
processes. Many such procedures and methods for preparing pharmaceutical
compositions
are known in the art, see for example L. Lachman et al. The Theory and
Practice of Industrial
Pharmacy, 4th Ed, 2013 (ISBN 8123922892).
The invention further relates to the following items, which may also be
formulated as claims:
Item 1: A tetrameric polypeptide comprising or consisting of
- a first polypeptide chain comprising a first ligand that binds to a HER2 D4
epitope, an
interdomain amino acid linker and a first immunoglobulin domain,
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- a second polypeptide chain comprising a second immunoglobulin domain,
wherein the first
immunoglobulin domain and the second immunoglobulin domain together constitute
a
second ligand, particularly an Fab domain, that binds to a HER2 D1 epitope,
- a third polypeptide chain comprising a third ligand that binds to a HER2
D4 epitope, an
interdomain amino acid linker and a third immunoglobulin domain, and
- a fourth polypeptide chain comprising a fourth immunoglobulin domain,
wherein the third
immunoglobulin domain and the fourth immunoglobulin domain together constitute
a fourth
ligand, particularly an Fab domain, that binds to a HER2 D1 epitope.
Item 2: The polypeptide according to item 1, wherein said first immunoglobulin
domain is
substantially the same as said third immunoglobulin domain, and/or wherein
said second
immunoglobulin domain is substantially the same as said fourth immunoglobulin
domain.
Item 3: The polypeptide according to item 1 or 2, wherein said first ligand is
substantially the
same as said third ligand.
Item 4: The polypeptide according to any one of the preceding items, wherein
said first ligand
and/or said third ligand comprises or consists of a single-chain variable
fragment polypeptide
chain comprising an scFv heavy chain, an scFv linker chain, and an scFv light
chain.
Item 5: The polypeptide according to item 4, wherein said scFv heavy chain is
the VH
domain of 4D5, and wherein said scFv light chain is the VL domain of 4D5.
Item 6: The polypeptide according to any one of the items 1 to 5, wherein
- said first immunoglobulin domain is a VL domain, and said second
immunoglobulin domain
is a VH domain and/or wherein said third immunoglobulin domain is a VL domain,
and said
fourth immunoglobulin domain is a VH domain;or
- said first immunoglobulin domain is a VH domain, and said second
immunoglobulin domain
is a VL domain and/or wherein said third immunoglobulin domain is a VH domain,
and said
fourth immunoglobulin domain is a VL domain.
Item 7: The polypeptide according to item 6, wherein said VH domain is C-
terminally linked to
a CH1 domain.
Item 8: The polypeptide according to item 7, wherein said CH1 domain is C-
terminally linked
to a CH2 domain or a CH2 domain and a CH3 domain.
Item 9: The polypeptide according to any one of the items 6 to 8, wherein said
VL domain is
C-terminally linked to a CL domain.
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Item 10: The polypeptide according to any one of the items 6 to 9, wherein
said VH domain is
selected from the VH domain of A21 and the VH domain of 702, and wherein said
VL
domain is selected from the VL domain of A21 and the VL domain of 702.
Item 11: The polypeptide according to any one of the preceding items, wherein
said
interdomain amino acid linker consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16,
17, 18, 19 or 20 amino acids.
Item 12: The polypeptide according to any one of the preceding items, wherein
said
interdomain amino acid linker comprises or consists of amino acids G, A, J, S,
T, P, C, V, M
and E, particularly wherein said interdomain amino acid linker comprises or
consists of amino
acids G, S, A and T.
Item 13: The polypeptide according to any one of the preceding items, wherein
said
interdomain amino acid linker is characterized by an amino acid sequence
(GGGGS)n, with n
being 1, 2, 3, 4 or 5.
Item 14: The bispecific HER2-polypeptide according to any one of the preceding
items,
wherein said interdomain amino acid linker comprises or is a sequence
characterized by one
of SEQ ID 17, SEQ ID 55 to SQ ID 69 and SEQ ID 82 to SEQ ID 91.
Item 15: The polypeptide according to any one of the preceding items, wherein
said
interdomain amino acid linker comprises or consists of a peptide sequence
selected from
one of SEQ ID 17, SEQ ID 55 to SQ ID 69 and SEQ ID 82 to SEQ ID 91 or a
functional
equivalent peptide sequence having a sequence identity of at least 70%.
Item 16: The polypeptide according to any one of the preceding items for use
in a method for
the prevention or treatment of a malignant neoplastic disease associated with
expression of
Her2.
Wherever alternatives for single separable features such as, for example, an
isotype protein
or coding sequence, ligand type or medical indication are laid out herein as
"embodiments", it
is to be understood that such alternatives may be combined freely to form
discrete
embodiments of the invention disclosed herein. Thus, any of the alternative
embodiments for
a detectable label may be combined with any of the alternative embodiments of
ligand and
these combinations may be combined with any medical indication or diagnostic
method
mentioned herein.
The invention is further illustrated by the following examples and figures,
from which further
embodiments and advantages can be drawn. These examples are meant to
illustrate the
invention but not to limit its scope.
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Description of the Figures
Fig. 1 shows schemes of biparatopic anti-HER2 binding agents.
Fig. 2 shows further schemes of biparatopic anti-HER2 binding agents
Fig. 3 shows a vector map of a plasmid for co-expression of the light
and heavy
chain of construct 441 in CHO cells (Pymex10 based vector with double
expression cassette [CMV GOI polyA]).
Fig. 4 shows a vector map of a plasmid for co-expression of the light
and heavy
chain of construct 4702 in CHO cells (Pymex10 based vector with double
expression cassette [CMV GOI polyA]).
Fig. 5 shows a vector map of a plasmid for expression of construct 841 in
CHO cells.
Fig. 6 shows an elution profile of construct 441 from Protein A
affinity
chromatography.
Fig. 7 shows an elution profile of construct 441 from cation exchange
chromatography.
Fig. 8 shows an elution profile of construct 441 from size exclusion
chromatography.
Fig. 9 shows a Coumassie-stained SDS-PAGE gel of fractions from
purification of
construct 441.
Fig. 10 shows the viability testing of CHOs during the expression of
construct 441
(scFV-IgG). Expression optimization of construct 441 in CHOs cells for
indicated time. Cells were cultured in CHOgro medium from Mlrus (MIR 6260)
and additionally fed with free cysteine (reduced form) (2), glutathione (3),
fetal
calf serum (4) or all additives respectively (5). CHO cells were analyzed on
CASY cell counter (Scharfe System).
Fig. 11 shows a Western blot of construct 441 expression, secreted to
the medium of
CHO cells after indicated times. Cells were cultured in CHOgro medium from
Mlrus (MIR 6260) (1) and additionally fed with free cysteine (reduced form)
(2), glutathione (3), fetal calf serum (4) or all additives together (5),
respectively. Protein was precipitated from medium by acetone precipitation
and re-solubilized in SDS PAGE buffer. Proteins were resolved on 4-12 %
gradient gel and the western blot was analyzed on an Odyssey system (LI-
COR). Purified intact full length construct 441 is shown as control (A) and
runs
above the 170 kDa marker. Molecular weight marker Page ruler from Thermo
Scientific is shown in red.
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Fig. 12 shows cell proliferation assays (XTT) with BT474 cells after 4
days of
treatment. Trastuzumab (TZB), biparatopic DARPin (6L1G) and different
fusion variants of the biparatopic construct. LF IgG HL (murine parent of
construct 441), HF IgG HL (murine parent of construct 241) show similar anti-
proliferative activity compared to the biparatopic DARPin 6L1G, which is
superior to trastuzumab (TZB). HF IgG LH (murine variant, no seq.) and LF
IgG LH (murine variant, no seq.) show reduced anti-proliferative activity
compared to biparatopic DARPin and higher 1050 concentrations.
Fig. 13 shows cell proliferation assays (XTT) with BT474 cells after 4
days of
treatment to test the effect of the linker length. Biparatopic DARPin (6L1G)
and different fusion linker variants of the biparatopic construct (murine
parent
construct of 441) are compared. The 2-AA linker (GS) shows highest anti-
proliferative activity. The 4-, 7- and 12-AA linkers show similar activity.
The 22-
AA linker variant shows reduced activity.
Fig. 14 shows cell proliferation assays (XTT) with BT474 cells after 4 days
of
treatment. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441),
biparatopic construct 411 (humanized kappa1 VH1) and biparatopic construct
443 (humanized kappa4 VH3). All show similar plateau levels of anti-
proliferative activity, except 443, which shows reduced activity.
Fig. 15 shows cell proliferation assays (XTT) with BT474 cells after 4 days
of
treatment with different humanized versions of A21 IgG, when fused to TZB
scFv. The strategy of humanization is described above. Different variants use
humanized kappa1 VH3 or a humanized kappa1 VH core graft.
Fig. 16 shows XTT cell proliferation assay with BT474 cells after 4
day of treatment.
Tetravalent IgG (HF IgG HL and LF IgG HL murine) versus bivalent Fab
fusions (HF Fab HL and LF Fab HL murine). All constructs show similar
plateau and I050 values.
Fig. 17 shows XTT cell proliferation assay with SKBR3 cells after 4
day of treatment.
Biparatopic DARPin (6G) biparatopic construct (441 if), trastuzumab (TZB).
Fig. 18 shows cell proliferation assays (XTT) with CALU-3 cells after 4
days of
treatment. Biparatopic DARPin (6G), biparatopic construct (construct 441
(441tf), trastuzumab (TZB).
Fig. 19 shows cell proliferation assays (XTT) with BT474 cells after 4
days of
treatment, testing effect of domain 1 binding unit. Biparatopic construct with
A21 (construct 441ff) or 702 fusions show different I050 and plateau level.

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Fig. 20 shows cell proliferation assays (XTT) with BT474 cells after 4
days of
treatment, testing the effect of domain 1 binding unit. Biparatopic construct
with A21 (construct 441) or with 39S (39s HF IgG H)L
Fig. 21 shows XTT cell proliferation assays with HCC1419 cells after 4
days of
treatment. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441tf)
and bivalent LF-oaFabFc (A21-TZB-40a). 441 and 6G show similar inhibition
of cell proliferation after 4 days. LF-oaFabFc show slightly reduced
inhibition
of cell proliferation compared to 441.
Fig. 22 shows XTT cell proliferation assay with BT474 and HCC1419
cells after 4 day
of treatment. All human.
Fig. 23 shows XTT cell proliferation assay with BT474 and HCC1419
cells after 4 day
of treatment. All human.
Fig. 24 shows a) in the upper panel XTT cell proliferation assays with
BT474 (left) and
HCC1419 (right) cells after 4 day of treatment; and in the lower panel XTT
cell
proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 day
of
treatment (variants with higher affinity (NGS and GGG)); b) repeated
experiments with a new expression of NGS.
Fig. 25 shows XTT cell proliferation assays with BT474 (left) and
HCC1419 (right)
cells after 4 day of treatment.
Fig. 26 shows XTT cell proliferation assays with HCC1419 cells grown as 3D
spheroids.
Fig. 27 shows Western Blots 24 hours post treatment (BT474) with
indicated agents
(murine).
Fig. 28 shows in the upper panel Induction of apoptosis in BT474 cells
after 3 days of
treatment. Average number of propidium iodide (PI) positive cells was
determined for 4 replicates, counted by cell profiler and was analyzed with
Student's t-test. Biparatopic construct (441, 441ff ) induced significantly
more
cell death than trastuzumab (TZB). 441 and biparatopic DARPin (6L1G) show
similar level of cell death; and in the lower panel Induction of apoptosis in
BT474 cells after 3 days of treatment. Average number of annexin-V positive
cells was determined for 3-4 replicates, counted by cell profiler and was
analyzed with Student's t-test. Biparatopic construct 441 induced
significantly
more apoptosis than trastuzumab (TZB). Construct 441 and 6L1G show
similar level of apoptosis.
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Fig. 29 shows images of BT474 cells treated with the indicated agents
for 3 days.
Fig. 30 shows Alexa647-labeled trastuzumab (TZB), biparatopic
construct 441 and
biparatopic one armed constructs oaLF and oaHF were incubated for 1 h at
100 nM concentration with 3 million BT474 cells in 3 ml PBS containing NaN3
(0.1%) and BSA (1 %) at 4 C. Note that BT474 cells were pre-treated with 0.1
% NaN3 in PBS with 1% BSA to block internalization before binding. Cells
were analyzed afterwards on CyFlow Space instrument (Partec). All binding
agents show specific binding to the surface of HER2-positive BT474 cells.
Fig. 31 shows the induction of cell death after treatment with 100 nM
of indicated
agents. BT474, N87, HCC1419 and SKBR3 cells were seeded 24 h before
treatment in 96 black clear-well microscopy plates (Nunc), continuously
treated for 3 days and stained with HOECHST-33342 (Invitrogen) for total
cells and with propidium iodide (Sigma) for membrane-permeable dead cells.
Cells were analyzed on a Lionheart FX Automated Microscope (BioTek
Instruments) and the number of propidium iodide and HOECHST-33342
positive cells was quantified with Gen5 software (BioTek Instruments). The
ratio of propidium iodide and HOECHST-33342 positive cells was calculated
for 3 biological replicates and the mean and SD is shown in the corresponding
column plots. Biparatopic binding agents (6L1G, 441, 841, LFoa, 241, 641,
HFoa, 7C2LF) binding to domain 1 and 4 of HER2 induce continuously more
dead cells than trastuzumab (TZB) or the combination of trastuzumab and
pertuzumab (TZB+PZB) in HER2-positive cancer cells.
Fig. 32 shows the half-life of construct 441 in the serum of NSG mice.
Drawn sera of
mice with previous 441 injections (3 mg/kg) were analyzed by sandwich
ELISA. 441 showed an alpha phase of around 4.3 hours, followed by a beta
phase of more than 45 hours.
Fig. 33 shows in-vivo activity of 441 on N87 xenografts in SCID beige
mice. After N87
tumors had reached 150 mm3 in size, mice were treated with eight injections of
441 (10 mg/kg) during four weeks. 441 lead to significant tumor size reduction
compared to untreated mice and TZB (10 mg/kg) or huA21G (10 mg/kg)
treated mice.
Fig. 34 shows representative microscopy images of BT-474 cells after
treatment for 2
h with trastuzumab (TZB), huA21G (A21), their combination, or 441, and non-
treated cells, either without or with addition of an anti-human primary
antibody,
as controls. Nuclei were stained using 2-(4-amidinopheny1)-1H-indo1-6-
carboximidamide (DAPI), antibodies were detected with an anti-human Fc
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antibody from goat, and lysosomal compartments using an anti-LAMP1
antibody.
Fig. 35 shows the result of a time-course treatment and subsequent
surface protein
internalization and degradation assay for the constructs 441 and 841, hA21G,
trastuzumab (TZB), the combination of trastuzumab and hA21G (TZB +
hA21G), pertuzumab (PZB), the combination of trastuzumab and pertuzumab
(TZB + PZB), and the inhibitor of HSP90, geldanamycin (GA).
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Sequence listings:
SEQ ID No. 1 ("441 polypeptide 1"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEI KGSDIVLTQSPDSLAVSLGERATI NCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLI
SWAFTRKSGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQYS NYPWTFGQGTKVEI KRT
VAAPSVFI FP PS DEQLKSGTASVKCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDS KD
STYSLSSTLTLS KADYE KH KVYACEVTHQG LSSPVTKS FN RG EC
SEQ ID No. 2 ("441 polypeptide 2"):
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG HI SSSYATSTY
NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 3 ("4702 polypeptide 1"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEI KGSDIVMTQSPDSLAVSLGERATI NCRASQSVSGSRFTYMHWYQQKPGQPPKLLI
KYASI LESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEI PPWTFGQGTKVEI KRT
VAAPSVFI FP PS DEQLKSGTASVKCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDS KD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 4 ("4702 polypeptide 2"):
EVQLVQSGAEVKKPGASVKVSCKASGYS FTGYWMNWVRQAPGQG LEWIGMI HPLDAEI RA
NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQVS
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LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSK
SEQ ID No. 5 ("841 polypeptide 2"):
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG HI SSSYATSTY
NQKFQG RVTFTVDTSSSTAYM ELSS LRSE DTAVYYCVRSG NYEEYAM DYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 6 ("8702 polypeptide 2"):
EVQLVQSGAEVKKPGASVKVSCKASGYS FTGYWMNWVRQAPGQG LEWIGMI HPLDAEI RA
NQKFRDRVTITVDTSTSTAYLE LSSLRSE DTAVYYCARGTYDGG FEYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 7 ("841Fc polypeptide 1"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEI KGSDIVLTQSPDSLAVSLGERATI NCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLI
SWAFTRKSGVPDRFSGSGSGTDFTLTI SSLQAEDVAVYYCQQYS NYPWTFGQGTKVEI KRT
VAAPSVFI FP PS DEQLKSGTASVKCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDS KD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGG
PSVFLFP PKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSRKEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 8 ("841Fc polypeptide 2"):
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG HI SSSYATSTY
NQKFQG RVTFTVDTSSSTAYM ELSS LRSE DTAVYYCVRSG NYEEYAM DYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPP KP KDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK

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SEQ ID No. 9 ("87C2Fc polypeptide 1"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEI KGSDIVMTQSPDSLAVSLGERATI NCRASQSVSGSRFTYMHWYQQKPGQPPKLLI
KYASI LESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEI PPWTFGQGTKVEI KRT
VAAPSVFI FP PS DEQLKSGTASVKCLLN N FYPREAKVQWKVDNALQSGNSQESVTEQDS KD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGG
PSVFLFP PKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSRKEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 10 ("87C2Fc polypeptide 2"):
EVQLVQSGAEVKKPGASVKVSCKASGYS FTGYWMNWVRQAPGQG LEWIGMI HPLDAEI RA
NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 11 ("241 polypeptide 1"):
DIVLTQSPDSLAVSLGERATI NCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS
GVPDRFSGSGSGTDFTLTI SS LQAEDVAVYYCQQYS NYPWTFGQGTKVE I KRTVAAPSVFI F
PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 12 ("241 polypeptide 2"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVE I KGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQGLEWMG H I
SSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
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FPAVLQSSGLYSLESVVTVPSSSLGTQTYI CNVN H KPSNTKVDKRVEPKSCDKTHTCP PCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 13 ("641 polypeptide 2"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVE I KGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG H I
SSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 14 ("scFy TZB L chain"):
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF
SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI K
SEQ ID No. 15 ("scFy TZB H chain"):
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID No. 16 ("scFy TZB linker"):
GGGGSGGGGSGGGGSGGGGS
SEQ ID No. 17 ("B1 to B2 linker (link1)"):
GS
SEQ ID No. 18 ("A21 L chain")
DIVLTQSPDSLAVSLGERATI NCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS
GVPDRFSGSGSGTDFTLTI SS LQAEDVAVYYCQQYS NYPWTFGQGTKVE I KRTVAAPSVFI F
PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
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SEQ ID No. 19 ("A21 H chain with Fc")
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG HI SSSYATSTY
NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 20 ("A21 H chain for Fab")
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG HI SSSYATSTY
NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 21: (õTZB CDR H1")
GFN I KDTYI H
SEQ ID No. 22: (õTZB CDR H2")
RIYPTNGYTRYADSVKG
SEQ ID No. 23: (õTZB CDR H3")
WGGDGFYAMDY
SEQ ID No. 24: (õTZB CDR L1")
RASQ DV N TAVA
SEQ ID No. 25: (õTZB CDR L2")
SASFLYS
SEQ ID No. 26: (õTZB CDR L3")
QQHYTTPPT
SEQ ID No. 27: (õA21 CDR H1")
GYSFTGYFI N
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SEQ ID No. 28: (õA21 CDR H2")
HISSSYATSTYNQKFQG
SEQ ID No. 29 ("A21 CDR H3")
SGNYEEYAMDY
SEQ ID No. 30: (õA21 CDR L1")
RSSQTLLYSNNQKNYLA
SEQ ID No. 31: (õA21 CDR L2")
WAFTRKS
SEQ ID No. 32: (õA21 CDR L3")
QQYSNYPWT
SEQ ID No. 33: (õ702 CDR H1")
GYSFTGYWMN
SEQ ID No. 34: (õ702 CDR H2")
MIHPLDAEIRANQKFR
SEQ ID No. 35: (õ702 CDR H3")
GTYDGGFEY
SEQ ID No. 36: (õ702 CDR L1")
RASQSVSGSRFTYMH
SEQ ID No. 37: (õ702 CDR L2")
YASILES
SEQ ID No. 38: (õ702 CDR L3")
QHSWEIPPWT
SEQ ID No. 39 ("A21 L chain Vi")
DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQQKPGQPPKLLISWAFTRKS
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF
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PPSDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 40: (õA21 H chain V1")
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVRQAPGQG LEWMG HI SSSYATSTY
NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 41: (õA21 L chain V2")
DIVLTQSPDSLAVSLGERATI NCRSSQPLEYSNNQWNYLAWYQKKPGQPPKLLISWAFTRK
SGVP DRFSGSGSGTDFTLTISSLQAEDVAVYYCGQYSDYPNTFGQGTKVEI KRTVAAPSVFI
FPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 42: (õA21 H chain V2")
QVQLVQSGAEVKKPGASVKVSCKASGYPFTQYFI HWVREAPGQG LEWMG HI SSSYATVDY
NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 43: (õscFv TZB L chain V1")
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF
SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI K
SEQ ID No. 44: (õscFv TZB H chain V1")
EVQLVESGGG LVQPGGS LRLSCAASG FN I KDTYI HWVRQAPG KG LEWVARIYPTNAYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGTGFYAMDYWGQGTLVTVSS
SEQ ID No. 45: (õAlternative Fc part - no mut")
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLP

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PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 46: (õAlternative Fc part - Knob into hole - knob site V1")
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 47: (õAlternative Fc part - Knob into hole - hole site V1")
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 48: (õAlternative Fc part - Knob into hole - knob site V2")
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLP
PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 49: (õAlternative Fc part - Knob into hole - hole site V2")
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVCTLP
PSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID No. 50: (õA21 L chain V3")
DIQLTQSPSSLSASVGDRVTITCRSSQTLLYSNNQKNYLAWYQQKPGKAPKLLISWAFTRKS
GVPSRFSGSGSGTDFTLTI SSLQPE DFATYYCQQYS NYPWTFGQGTKVE I KRTVAAPSVFI F
PPSDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 51: (õA21 H chain V3")
EVQLVQSGPELVQPGGSVRISCAASGYSFTGYFI NWVKQAPG KG LEWISH I SSSYATSTYN
QSFKGRATFSVDTSSSTAYMQLNSLRAEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSSA
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STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 52: (õA21 H chain V4")
VQLVESGGGLVQPGGSLRLSCAASGYSFTGYFINWVRQAPGKGLEWVSHISSSYATSTYN
QSVKGRFTFSVDTSSSTAYLQMNSLRAEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
SEQ ID No. 53: (õTZB H chain V2")
QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEQMGRIYPTNGYTRY
DPKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID No. 54: (õTZB H chain V3")
EVQLVQSGPELVQPGGSLRLSCAASGFNIKDTYIHWVKQAPGKGLEWISRIYPTNGYTRYD
PSFKGRATISADTSSNTAYLQVNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID No. 55: (õPoly-Gly-linker")
GGGGG
SEQ ID No. 56: (õOva-linker")
GSGSGS
SEQ ID No. 57: (õTrans-linker")
GSGGGTGGGSG
SEQ ID No. 58: (õPro-linker")
PPP
SEQ ID No. 59: (õPAS-linker 1")
ASPAAPAPASPAAPAPSAPAA
SEQ ID No. 60: (õPAS-linker 2")
ASAAAPAAASAAASAPSAAAA
SEQ ID No. 61: (õPAS-linker 3")
AASPAAPSAPPAAASPAAPSAPPAA
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SEQ ID No. 62: (õPAS-linker 4")
ASPASA
SEQ ID No. 63: (õPAS-linker 5")
AS PAS PASA
SEQ ID No. 64: (,,XS-linker 1")
PAGSP
SEQ ID No. 65: (õXS-linker 2")
STEPS
SEQ ID No. 66: (,,XS-linker 3")
STEEG
SEQ ID No. 67: (õXS-linker 4")
GSAPG
SEQ ID No. 68: (õProper-linker1")
GASTP
SEQ ID No. 69: (õProper-linker2")
GPSAT
SEQ ID No. 70: (õ39s light chain")
DIVMTQTPLSLSVTPGQPASISCKSSQSVFFRSN N KN I LAWYLQKPGQPPQLLIYWASSRES
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYFGSPFTFGPGTKVDIKRTVAAPSVF1 F
PPSDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 71: (õ39s heavy chain")
EVQLVESGGG LVKPGGSLRLSCAASG FTFSSYS MSWVRQAPG KG LEWVSSI SSSSSYIYYA
DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDAYNYYYFDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
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VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 72: (õH218 light chain")
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVS
N RFSGSKSG NTAS LTI SG LQAE DEADYYCSSYTSSSTLVFGGGTKLTVLGTVAAPSVFI FPP
SDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 73: ("H218 heavy chain")
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGQGLEWMGWISAYNGNTN
YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREGDGAFDYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSK
SEQ ID No. 74: (õMF3958 light chain")
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEI KRTVAAPSVFI FP PSDEQL
KSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 75: (õMF3958 heavy chain")
QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYI NWVRQAPGQGLEWIGRIYPGSGYTSYA
QKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPPVYYDSAWFAYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 76: (õA21 L chain V4")
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DIVLTQSPDSLAVSLGERATI NCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS
GVPDRFSGSGSGTDFTLTI SS LQAEDVAVYYCQQYS NYPWTFGQGTKVE I KRTVAAPSVFI F
PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 77: (õA21 H chain with Fc V2")
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQG LEWMG HI SSSYATSTY
NQKFQG RVTFTVDTSSSTAYM ELSS LRSE DTAVYYCVRSG NYEEYAM DYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPP KP KDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVM H EALH N HYTQKS LS LSK
SEQ ID No. 78: (õ702 light chain")
DIVMTQSPDSLAVSLGERATI NCRASQSVSGSRFTYMHWYQQKPGQPPKLLI KYASI LESGV
PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEI P PWTFGQGTKVEI KRTVAAPSVFI FP
PSDEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No. 79: (õ702 heavy chain")
EVQLVQSGAEVKKPGASVKVSCKASGYS FTGYWMNWVRQAPGQG LEWIGMI HPLDAEI RA
NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTP EVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSN KALPAP I EKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSK
SEQ ID No. 80: (õTZB-HC")
EVQLVESGGGLVQPGGSLRLSCAASGFN I KDTYI HWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPP KP KDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP REEQYNSTYR

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VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSK
SEQ ID No. 81: (õTZB-LC")
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF
SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID 82 ("GS-linker 1"): GGGGS
SEQ ID 83 ("GS-linker 2"): GGGGSGGGGS
SEQ ID 84 ("GS-linker 3"): GGGGSGGGGSGGGGS
SEQ ID 85 ("GS-linker 4"): GGGGSGGGGSGGGGSGGGGS
SEQ ID 86 ("GS-linker 5"): GGGGSGGGGSGGGGSGGGGSGGGGS
SEQ ID 87 ("GA-linker "): GAAGAAG
SEQ ID 88 ("GT-linker"): GGTGGT
SEQ ID 89 ("ST-linker"): STSTS
SEQ ID 90 ("PA-linker"): PAPAP
SEQ ID 91 ("SA-linker"): SAASAAS
SEQ ID 92 ("HF-oaFabFc light chain" / "A21-TZB-2oa light chain"):
DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK
SEQ ID 93 ("HF-oaFabFc heavy chain" / "A21-TZB-2oa heavy chain"):
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ
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KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVE I KGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFI NWVREAPGQGLEWMG H I
SSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLESVVTVPSSSLGTQTYI CNVN H KPSNTKVDKRVEPKSCDKTHTCP PCP
APELLGGPSVFLFPP KP KDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLP
PSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTP PVLKSDGSFFLYSKLTVD
KSRWQQG NVFSCSVM H EALH N HYTQKS LS LS PG K
Example 1: Biparatopic Anti-HER2 Binding Agents
The inventors have generated biparatopic IgG derivatives. In contrast to other
available
biparatopic HER2-targeting antibodies, e.g. the antibody-drug conjugate (ADC)
from
Medimmune MEDI4276 (Li et al., 2016), these IgGs show very strong anti-tumor
activity as
"naked" binding proteins, i.e., without attached drug (Kast et al., in
preparation). Thus, it is
believed that these novel biparatopic anti-HER2 IgGs combine the mechanisms of
action of
trastuzumab plus pertuzumab plus the action of small molecule kinases
inhibitors against
HER2 in one single molecule. In addition, potential off-target effects of the
biparatopic anti-
HER2 IgGs are expected to remain far below those of ADC fusions, such as T-DM1
or
MEDI4276, as they can only act on HER2-addicted cells, while ADCs can via
their toxin act
in many healthy tissue. This opens up the therapeutic windows for new
combination
therapies. Furthermore, pan-ErbB inhibition by polymerization of HER2
receptors may
passively block compensatory activation of other receptor tyrosine kinases
(RTKs). The
biparatopic anti-HER2 binding agents interfere with the free lateral movement
of HER2
.. receptors on the cell surface of HER2-amplified cancer, yet without
inducing signaling
competent complexes, which may block the activation of other RTKs.
Consequently,
biparatopic anti-HER2 binding agents may show strong synergies with small
molecule
inhibitors, which tend to induce expression of compensatory RTKs that
eventually drives
escape from therapy. Therefore, biparatopic anti-HER2 IgGs bear a very high
potential to
elicit strong anti-tumor synergies in combination with small-molecule
inhibitors on a broad
panel of HER2-amplified cancers. The potential for synergies with small-
molecule inhibitors
is superior to current single-specificity antibodies or antibody combinations.
Illustrative schemes of preferred biparatopic IgG constructs are shown in Fig.
1 and 2
Data regarding preparation, and biological activity of the biparatopic IgG
constructs are
shown in Figs 3 to 26.
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Protocol for Production of Biparatopic IdGs in CHOs cells
Vector design
Bicistronic plasmids containing two expression cassettes or two vector systems
were
constructed for co-transfections. Derivatives of plasmid pYMex10 (Morphosys)
were used for
the bicistronic strategy. In the resulting plasmid constructs, the coding
sequences of the
polypeptide chains of the multimeric constructs were each under the control of
an individual
CMV promotor and terminated by a polyA tail signal as for example taken from
bovine growth
hormone or simian virus 40 (see Fig. 3 and 4). pcDNA 3.1 (Thermo) derived
vectors were
used for co-transfections. Here, the individual polypeptide chains are on a
separate plasmid
reducing the risk of recombination of homologous elements and further allow to
adjust molar
ratios of plasmids for the transfections to improve the yield. Exchange of
genes of interest is
possible by standard cloning techniques. Examples of the resulting constructs
are depicted in
Fig. 5.
Expression in CHO-S
Exponentially growing CHO-S cells (Thermo) were seeded in CHOgro (Mirrus) at a
density of
four millions per ml in TPP600 bioreactors. Per ml of culture 3 pg of linear
polyethylenimine
(MW 25,000, PolySciences Inc) and 1.25 pg of highly pure plasmid DNA were
added with in-
between mixing. Eventually, cultures were supplemented with valproic acid to a
final
concentration of 1 mM. Proteins were expressed at 31 or 37 C, 8% CO2 and 180
rpm with a
50 mm throw in Kuhner ISF1-X shaker for up to 12 days.
Purification of molecules
Expression cultures were harvested by centrifugation at 1400 rpm and 4 C for
30 min.
Supernatants were furthermore cleared by 0.22 pm filtration and adjusted to pH
of 7. All
subsequent purification steps were performed at 4 C. Supernatants were applied
to PBS
equilibrated rProtein A columns (GE) operated on a AEKTA Pure system. After
PBS wash,
bound protein was eluted by 0.1 M Glycine pH 2.75 (Chromatogram Fig. 6).
Fractions of
interest were pooled and buffer exchanged to 20 mM Bis/Tris methane 20 mM NaCI
pH 6.75.
Filtered protein solutions were loaded on Resource S (GE) columns and eluted
with a
gradient to 100% 20 mM Bis/Tris methane 1000 mM NaCI pH 6.75 (see Fig. 7).
Desired
fractions were pooled and if required polished on SEC (5uperdex200 columns of
adequate
size; GE) for high purity (Fig. 8). Monomeric fractions were pooled, filtered,
and purity
analyzed on SDS page (Fig. 9). Eventually, pure protein was used for
biochemical and cell
assays as well as in-vivo studies.
HER2-related effects of tetravalent biparatopic IgGs
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The tetrameric (tetravalent and biparatopic) polypeptide constructs 441 and
4702 were
tested in various assays, which are all well-known to the skilled person, and
compared to the
tetravalent IgG-fusion MEDI4276 (without a toxin), the dimeric bivalent
biparatopic
polypeptide constructs 841, 8702 and Fc fusions thereof, the bivalent
biparatopic DARPin
construct 6L1G (see patent application WO 2014/060365 Al), single IgGs (TZB,
PZB, A21
and 702) and combinations thereof.
Growth inhibition was tested in an XTT cell viability assay using live-cell
high-content
microscopy, Hoechst staining and cell count. As shown in Table 1, constructs
441, 4702,
841, 8702 and the Fc fusions of 841 and 8702, 6L1G and the combination of TZB
with A21
resulted in full growth inhibition, whereas single antibodies and TZB combined
with PZB lead
to partial inhibition. Surprisingly, in the absence of a cytotoxic drug, the
antibody MEDI4276
stimulated growth of XTT cells.
The effect of the constructs on apoptosis/cell death were analyzed by live-
cell high-content
microscopy with annexin-V and PI staining or by detecting cleaved PARP in cell
lysates by
Western blot for analysis of PARP cleavage.
Constructs 441, 4702, 841, 8702 and their Fc fusions, 6L1G had an effect on
apoptosis,
whereas MEDI4276, single antibodies did not influence apoptosis, and TZB and
A21 had a
partial effect. The combination TZB+PZB is able to induce apoptosis in a very
small fraction
of cells or in fragile cell lines.
HER2 crosslinking on the cell surface, also termed "lockdown", was measured by
fluorescence recovery after photobleaching (FRAP) and single cell localization
microscopy. A
reduction of the FRAP signal indicates lower mobility of cells and therefore
crosslinking in
response to the polypeptide constructs.
441, 4702 and 6L1G resulted in lockdown of receptors, and partial crosslinking
effect was
measured for 841, 8702 and their Fc fusions as well as the antibody
combinations TZB+PZB
and TZB+A21. Single antibodies had no effect on crosslinking.
Furthermore, HER2 internalization into cells was analyzed by a surface protein
internalization
and degradation assay, confocal microscopy and flow cytometry as described in
detail in
example 2.
The tetravalent biparatopic constructs 441, 4702 and MEDI4276 displayed a
strong effect on
HER2 internalization, whereas a recycling inhibition was detected for the
combinations
TZB+PZB and TZB+A21). The remaining constructs showed no effect.
HER2 degradation was tested by a surface protein internalization and
degradation assay and
Western blot detection of total HER2. Here, 441 and 4702 lead to rapid strong
degradation.
MEDI4276 had an effect on degradation, but less strong compared to 441 and
4702. In
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contrast, the antibody combinations resulted in slow degradation and the
remaining
constructs had no effect.
Construct Growth Apoptosis/ "Lockdown" HER2 HER2
inhibition cell death (crosslinking
Internalization Degradation
on cells)
441/4702 Yes Yes Yes Strong
Rapid,
Strong
MEDI4276 No No Not Same as 441
Less than
(without (stimulating determined but 441
toxin) !) expected
841/8702 Yes Yes Partial No No
and Fc
fusions
thereof
6L1G Yes Yes Yes No No
Single Partial No No No No
antibodies
TZB+PZB Partial No (yes) Crosslinking Recycling
Very slow
inhibition (very
weak)
TZB+A21 Yes Partial Crosslinking
Recycling Slow
inhibition
Table 1: Results of assays measuring HER2-related effects of polypeptide
constructs
Table 2 shows the results of HER2 binding studies performed with the same
constructs as
the experiments described above. Binding was determined by flow cytometry and
additionally
by size-exclusion chromatography/multi angle light scattering (SEC-MALS) in
case of
complex formation between the biparatopic IgG constructs and HER2.
All constructs bound the extracellular domains 1, 2 and/or 4 as expected (see
Table 2). 441,
4702 and the combination TZB + A21 displayed an extremely slow off rate which
was slower
compared to the remaining constructs.
Interestingly, all biparatopic constructs resulted in a HER2 binding
stoichiometry of close to
1:1. Single antibodies and antibody combinations only lead approximately to a
1:2
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Serum half-life of construct 441 was further tested by intra venous injection
in NSG mice and
time resolved detection of biparatopic IgG in blood samples by ELISA. To
determine the half-
life of 441, 3 mg/kg of purified construct were intravenously injected in NSG
mice. At
indicated time points mice were bleed, whole blood allowed to clot and sera
gained by taking
supernatants of centrifuged samples. A standard capture ELISA was used to
evaluate serum
levels of 441 (Fig. 32). Anti-human Fc antibody from mouse was directly coated
on maxisorb
plates. Bound 441 and sera spiked 441 standards were revealed by anti-kappa
chain
antibodies from goat conjugated to alkaline phosphatase. Data was fitted with
a two-phase
decay model and resulting half-life was calculated to be 4.3+45.3 hours (a and
13 phase).
Construct Binds to Off Rate Bound Total # Serum Half Molecule
Complex
HER2 Paratope life r weight
size on
molecules per kDa
cells (single
on average molecule
molecule)
in solution
441/4702 HER2 extremely Close to 1:1 4 45 hours
200 Large
ECD1/4 slow
2.7 x 10A-4
MEDI4276 HER2 Not Close to 1:1 4 rapid 200
Not
(without toxin)
ECD2/4 determined clearance
determined
But (app. 1-3
day)
expected
extremely
slow
841/8702 and HER2 Very slow Close to 1:1 2 Not
72 (+FC = Not
Fc fusions ECD1/4 determined 122)
determined
thereof
6L1G HER2 Very slow Close to 1:1 2 5 min (12
32 Medium
ECD1/4 hours
PEGylated)
Single HER2 Very slow Close to 1:2 2 12 days
145 Small
antibodies
ECD1/2/4
TZB+PZB HER2 Very slow Close to 1:2 2 12 days 145
Not
ECD2/4
determined
TZB+A21 HER2 extremely Close to 1:2 2 12 days 145
Not
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ECD1/4 slow
determined
3.66 x 10A-4
Table 2: HER2 binding properties and other parameters of polypeptide
constructs
Surprisingly, the tetrameric tetravalent biparatopic constructs 441 and 4702
lead to strong
inhibition of cell proliferation, induced cell death by apoptosis and led to
crosslinking of HER2
on the cell surface and induced strong HER2 internalization and strong HER2
degradation in
addition to their excellent binding properties to HER2. 441 and 4702 were
superior to or
scored equally well as all other constructs in all categories.
TZB+PZB and TZB+A21 resulted in a decrease of total HER2 (very weak in case of
TZB+PZB) which was attributed to recycling inhibition, a mechanism by which
HER2 is
degraded without prior intracellular accumulation (see Fig. 35).
To determine the effect of construct 441 on tumor growth, SCID beige (Charles
River) mice
were inoculated on the right flank with five million N87 cells in 50% matrigel
(Corning). After
tumors had reached around 150 mm3 mice were treated with 10 mg/kg 441 for
eight times
with a three to four-day interval. Treated mice responded to 441 with tumor
burden reduction.
Growth arrest was initially seen for TZB (10 mg/kg) and hA21G (10 mg/kg)
treated mice and
tumors of control mice (labeled 'PBS' in Fig. 33) showed unhindered
progression (Fig. 33).
Example 2: Enhanced Internalization, Lysosomal Trafficking, and Degradation of
HER2 by
revealed molecule 441
Microscopy with BT-474 and HCC1419 breast cancer cells
For microscopy of fixed samples, cells were seeded at a density of 4.104 cm-2
in p-slides
(Ibidi, cat. no. 80824) in complete medium. On the next day, cells were
treated with the
respective molecules. After 2 h, cells were once washed with Dulbecco's
phosphate buffered
saline (DPBS), and fixed by addition of 4% (w/v) paraformaldehyde dissolved in
DPBS and
incubation at room temperature for 10 min. Next, cells were washed twice with
PBSBA+T
(DPBS supplemented with 1% (w/v) bovine serum albumin (BSA), 0.1% (w/v) sodium
azide,
and 0.5% (w/v) Tween-20). Afterwards, cells were incubated in anti-LAMP
antibody (Cell
Signaling Technology, cat. no. D4015) dissolved at 1:150 (v/v) in PBSBA+T,
further
supplemented with 100 ng m1-1 2-(4-amidinopheny1)-1H-indo1-6-carboximidamide
(DAPI) for
min at room temperature. Cells were then washed twice with PBSBA+T, and
30 subsequently anti-mouse, conjugated to Alexa Fluor 488 (Thermo Fisher
Scientific, cat. no.
A11001) and anti-human, conjugated to Alexa Fluor 647 (Thermo Fisher
Scientific, cat. no.
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A-21445) antibodies from goat, dissolved in PBSBA+T, were added and incubated
for 30 min
at room temperature. Next, cells were washed twice with PBSBA+T, fixed once
more by
addition of 4% (w/v) paraformaldehyde dissolved in DPBS and incubation at room
temperature for 10 min, finally washed once with PBSA, and stored in PBSA
(DPBS
supplemented with 0.1% (w/v) sodium azide) at 4 C until measurement. Imaging
was
performed on a SP5 confocal laser scanning microscope (Leica). The images show
that for
both cell lines, only 441 was able to induce its rapid internalization, and
shows strong
colocalization with lysosomal (LAMP1-positive) compartments.
Surface protein internalization and degradation assay
To quantify internalization and degradation of HER2 upon treatments, we
performed a
quantitative surface protein internalization and degradation assay was
performed. In brief, a
stable Flp-In TREx HEK293 cell line (Thermo Fisher Scientific, cat. no.
K650001) was
generated according to the instructions of the manufacturer, in which a
HaloTag-HER2
receptor fusion can be overexpressed upon induction. For the assay, cells were
seeded two
days before the first treatment, and one day before treatment, doxycycline was
added to
induce stable overexpression for 24 h. Treatments (100 nM) were added at
indicated time
points, referring to the time of cell labeling.
After completion of the treatment time intervals, cells were labeled in a two-
step procedure. A
HaloTag ligand containing to Alexa Fluor 660 (HTL-AF660, Promega, cat. no.
G8472), which
.. is completely cell-impermeable and therefore stains surface receptors only,
was coupled in a
first labeling step. A cell-permeable HaloTag ligand containing tetramethyl
rhodamine (HTL-
TMR, Promega, cat. no. G8252), was, in the second step, applied to stain all
receptor fusion,
which resides in intracellular compartments. Thus, signals originating from
surface and
internal receptor are detected in separate channels on a flow cytometer.
Therefore,
information regarding the localization, and using the rescaling procedure
described below,
about the quantitative distribution, can be obtained. A commercially available
dead-cell stain
was used for exclusion of permeabilized (dead) cells from analysis, for which
all receptor
would appear to be on the surface. Fluorescence intensities in each channel
for 2000-10000
cells was recorded using a LSR ll Fortessa (BD), and single, non-permeabilized
cells gated.
Mean fluorescence intensities of these populations were obtained using FlowJo
10.4
(FlowJo).
Data processing
To correct for different detection efficiencies of the flow cytometry
instrument in the channels
for AF660 and TMR, the data were scaled, yielding relative abundances. A
control sample
(utr.,s.), in which the first (HTL-AF660-labeling) step is omitted, is
required to this end. In this
sample, all HaloTag molecules will react with HTL-TMR in this "single" (s.)
labeling
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procedure, irrespective of their localization. Using the mean fluorescence
intensities (MFI) of
the singlet, non-permeabilized cell population, the normalized (feature-
scaled) signal STmR in
the TMR channel for a samples is obtained by normalizing to a single-labeled,
untreated
control sample (utr.,s.) and background subtraction:
MF/TmR (sample) ¨ MF/TmR(utr.,unlab.)
STmR(sample) = _____________________________________________ #(eq.
MF/TmR(utr., s.) ¨ MF/TmR(utr.,unlab.)
where utr.,unlab. represents an untreated, unlabeled control (showing only
autofluorescence).
The first, surface-labeling step with cell-impermeable dye is virtually
saturating in the
complete two-step (double) labeling procedure. The normalized surface signal
SAF660 can
thus be defined in:
MF/AF66o (sample) ¨ MFIAF660(utr.,unlab.)
SAF660 (sample) =
#(eq. S2).
MF/AF66o(utr., d.) ¨ MFIAF660(utr.,unlab.)
The signal of a sample can be related to the surface signal from a double-
labeled control
(utr.,d.) and does not require a separate single-stained sample, however,
because internal
receptor is not accessible to HTL-AF660 and thus cannot be stained.
ASTmR, the difference from the single to the double labeling procedure in the
TMR channel,
now exactly corresponds to the number of molecules, which were blocked by the
first,
surface-specific step. The signal in the AF660 channel in the same double
labeling
experiment also is a direct correlate of this number of molecules:
STMR = STmR(Utr.,S.) STMR (utr., d.) = _AF66o,scaied (utr.,d.)#(eq. S3).
A correction factor CA can thus be defined, which relates the measured
intensity
SAF660(utr.,d.) (recorded in the AF660 channel) to SAF660,scaied(utr.,d.) (in
the scale of the TMR
channel):
SAF660,scaled (utr.,d.) = -AF660 (utr.,d.) x CA#(eq. S4).
Using signals from single-labeled and double-labeled, untreated cells,
calculation of CA is
possible as follows:
S-RiR _________________ (Utr.,S.) STMR(Utr.,d.) __ 100% ¨ STmR(Utr.,d.)
CA = #(eq. SS)
3AF660 (utrõ d.) SAF660 (utr., d.)
taking into consideration that the TMR signal of single-labeled, untreated
cells was, using eq.
Si, scaled to 100% before. Correction of the signals recorded in the AF660
channel, can, for
treated samples, now be done according to:
SAF660,scaled (tre.,d.) = -A x C - S AF660 (tre.,d.)#(eq. S6)
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STmOre.,d.) and SAF660,scaied(tre.,d.) then truly represent the abundance of
internal and
surface protein, respectively, and the sum
SAF660,scaied(tre.,d.)+SrmR(tre.,d.) represents the
amount of total protein, for a double-labeled, treated sample, always relative
to an untreated
control sample.
Data scaling as described above was performed and the results plotting using
MATLAB
R2017b (MathWorks), R 3.5.1, Prism 6.07 (GraphPad), and Exce12016 (Microsoft).
The
results are shown in Fig. 35. In contrast to all other constructs, construct
441 showed almost
HER2 internalization after less than 5 minutes.
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