Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/195092
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1
FERRITIN VARIANTS WITH INCREASED STABILITY AND COMPLEXATION
ABILITY
The present invention relates to new ferritin variants, wherein at least one
lysine residue is
deleted or substituted with a non-basic amino acid. The invention further
relates to a complex of
this polypeptide and a compound, a label or drug, and an isolated delivery
system comprising the
polypeptide or the complex of the invention (alone or as cellular system) as
well as uses of such
system for prophylaxis, therapy or diagnosis, in particular for therapy of
cancer or inflammatory
diseases.
BACKGROUND OF THE INVENTION
Ferritin has emerged as a promising protein-based nanocage thanks to its
unique
architecture, surface properties and high biocompatibility. However, to date,
no successful ferritin
drug complexes have reached the clinic (Truffi M et al., Pharmacol Res. 2016
May; 107:57-65).
The inventors surprisingly discovered that by deletion or substitution of
cysteine residues, several
properties of ferritin polypeptides can be significantly improved. One or more
problems described
in the prior art are solved by the ferritin variant polypeptides defined in
the appended claims and
in the second aspect of the invention described below. The described mutations
(i.e., deletions or
substitutions of lysine residues) were found to improve the properties of
ferritins as carriers, in
particular as nanocages for drugs and/or labels. The inventive ferritin
variant polypeptides provide
inter alia for: (i) improved encapsulation efficiency of drugs or labels into
ferritin nanocages; (ii)
improved nucleic acid binding properties of ferritin; (iii) improved stability
of ferritin nanocages;
(iv) improved stability of drug encapsulation in ferritin nanocages; (v)
improved protein recovery
after loading of ferritin nanocages; (vi) enhanced cytotoxicity against tumor
cells by drugs
enclosed within ferritin nanocages or attached to ferritin; (vii) improved
chemico-physical
properties of ferritin nanocages; (viii) decreased tendency of dimer formation
of ferritin
nanocages; (ix) decreased tendency of aggregation of ferritin nanocages.
SUMMARY OF THE INVENTION
In a first aspect the present invention relates to a polypeptide comprising a
transferrin
receptor binding domain (TRBD) of a ferritin variant wherein within the TRBD
the ferritin variant
in comparison to the wild-type ferritin on which it is based comprises one or
more glutamine
residues mutated into glutamic acid residues and/or one or more asparagine
residues mutated into
aspartic acid residues.
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In a second aspect the present invention relates to a ferritin variant
polypeptide, wherein at
least one, at least two, at least three or at least four, preferably four,
lysine residues, preferably
lysine residues at position 54, 72, 87 and/or 144 indicated with respect to
SEQ ID NO. 1 (human
wild-type heavy chain ferritin), are deleted or substituted with a non-basic
amino acid.
In a third aspect the present invention relates to a ferritin variant
polypeptide, wherein one
or more cysteine residues, in particular cysteine residues at position 91, 103
and/or 131 indicated
with respect to SEQ ID NO. 1, are deleted or substituted, preferably
substituted with serine
residues.
In a fourth aspect the present invention relates to a nucleic acid encoding
the polypeptide of
the first, second or third aspect.
In another aspect the present invention relates to a vector comprising the
nucleic acid of the
fourth aspect.
In a fifth aspect the present invention relates to a conjugate comprising the
polypeptide of
the first, second or third aspect and at least one label and/or at least one
drug
In a sixth aspect the present invention relates to a complex comprising at
least one
polypeptide of the first, second or third aspect and/or at least one conjugate
of the fifth aspect of
the present invention.
In a seventh aspect the present invention relates to an isolated targeted
delivery system
comprising a cell, wherein the cell comprises the polypeptide of the first
aspect, second or third,
the conjugate of the fifth aspect, or the complex of the sixth aspect of the
present invention.
In an eighth aspect the present invention relates to a pharmaceutical or
diagnostic
composition comprising the polypeptide of the first, second or third aspect,
the conjugate of the
fifth aspect, the complex of the sixth aspect or the isolated targeted
delivery system of the seventh
aspect and a pharmaceutically acceptable carrier and/or suitable excipient(s).
In a ninth aspect the present invention relates to the polypeptide of the
first, second or third
aspect, the conjugate of the fifth aspect, the complex of the sixth aspect,
the isolated targeted
delivery system of the seventh aspect for use in medicine.
In a tenth aspect the present invention relates to the polypeptide of the
first, second or third
aspect, the conjugate of the fifth aspect, the complex of the sixth aspect,
the isolated targeted
delivery system of the seventh aspect, or the pharmaceutical or diagnostic
composition of the
eighth aspect for use in treating, preventing or diagnosing a tumour,
preferably a solid tumour
and/or its metastases, preferably breast cancer, pancreatic cancer, bladder
cancer, lung cancer,
colon cancer, ovarian cancer, liver cancer, glioma/glioblastoma or a tumour
having hypoxic areas;
an inflammatory disease or ischemic areas, in particular in skin wounds or
after organ infarctus
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(heart) or ischemic retina; or for prophylactic or therapeutic vaccination, in
particular to prevent
or treat an infectious disease or cancer.
In an eleventh aspect the present invention relates to a method of treating,
preventing or
diagnosing a tumour, preferably a solid tumour and/or its metastases,
preferably breast cancer,
pancreatic cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer,
liver cancer,
glioma/glioblastoma or a tumour having hypoxic areas; an inflammatory disease
or ischemic areas,
in particular in skin wounds or after organ infarctus (heart) or ischemic
retina; or a method of
prophylactic or therapeutic vaccination, in particular to prevent or treat an
infectious disease or
cancer by administering an effective amount of the polypeptide of the first,
second or third aspect,
the conjugate of the fifth aspect, the complex of the sixth aspect or the
isolated targeted delivery
system of the eighth aspect to a subject in need thereof.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Before the present invention is described in detail below, it is to be
understood that this
invention is not limited to the particular methodology, protocols and reagents
described herein as
these may vary. It is also to be understood that the terminology used herein
is for the purpose of
describing particular embodiments only, and is not intended to limit the scope
of the present
invention, which will be limited only by the appended claims. Unless defined
otherwise, all
technical and scientific terms used herein have the same meanings as commonly
understood by
one of ordinary skill in the art.
The terms "peptide" or "polypeptide are used interchangeably in the context of
the present
invention to refer to a chain of at least two amino acids linked by peptide
bonds. Thus, the term
"polypeptide" in the context of the present invention is also used to refer to
amino acid chains with
more than 50, more than 100 or more than 150 amino acids.
The term "amino acid" encompasses naturally occurring amino acids as well as
amino acid
derivatives. In the context of the present specification, amino acids are
identified either using the
1-letter code (Hausman RE, Cooper GM (2004) or the 3-letter code . The cell: a
molecular
approach. Washington, D.C: ASM Press. p. 51. ISBN 978-0-87893-214-6). An amino
acid
identified with the letter X corresponds to any amino acid. An amino acid
identified with the letter
B corresponds to either D (asparagine) or N (aspartic acid). An amino acid
identified with the letter
Z corresponds to either E (glutamine) or Q (glutamic acid).
The terms "polynucleotide" and "nucleic acid" are used interchangeably herein
and are
understood as a polymeric or oligomeric macromolecule made from nucleotide
monomers.
Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as
but not limited
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to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a
polynucleotide is
formed through phosphodiester bonds between the individual nucleotide
monomers. In the context
of the present invention referred to nucleic acid molecules include but are
not limited to ribonucleic
acid (RNA) and its various forms (e.g. but not limited to ssRNA, LNA etc.),
deoxyribonucleic acid
(DNA), and mixtures thereof such as e.g. RNA-DNA hybrids. The nucleic acids,
can e.g. be
synthesized chemically, e.g. in accordance with the phosphotriester method
(see, for example,
Uhlmann, E. &Peyman, A. (1990) Chemical Reviews, 90, 543-584). "Aptamers" are
nucleic acids
which bind with high affinity to a polypeptide. Aptamers can be isolated by
selection methods
such as SELEmir146-a (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50; Klug
and Famulok
(1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981) from a large pool of
different single-
stranded RNA molecules. Aptamers can also be synthesized and selected in their
mirror-image
form, for example as the L-ribonucleoti de (Nolte et al. (1996) Nat.
Biotechnol., 14, 1116-9;
Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). Forms which have been
isolated in this
way enjoy the advantage that they are not degraded by naturally occurring
ribonucl eases and,
therefore, possess greater stability.
The term "identity" is used throughout the specification with regard to
polypeptide and
nucleotide sequence comparisons. In case where two sequences are compared and
the reference
sequence is not specified in comparison to which the sequence identity
percentage is to be
calculated, the sequence identity is to be calculated with reference to the
longer of the two
sequences to be compared, if not specifically indicated otherwise. If the
reference sequence is
indicated, the sequence identity is determined on the basis of the full length
of the reference
sequence indicated by SEQ ID, if not specifically indicated otherwise. For
example, a polypeptide
sequence consisting of 200 amino acids compared to a reference 300 amino acid
long polypeptide
sequence may exhibit a maximum percentage of sequence identity of 66.6 %
(200/300) while a
sequence with a length of 150 amino acids may exhibit a maximum percentage of
sequence
identity of 50 % (150/300). If 15 out of those 150 amino acids are different
from the respective
amino acids of the 300 amino acid long reference sequence, the level of
sequence identity
decreases to 45 %. The similarity of nucleotide and amino acid sequences, i.e.
the percentage of
sequence identity, can be determined via sequence alignments. Such alignments
can be carried out
with several art-known algorithms, preferably with the mathematical algorithm
of Karlin and
Altschul (Karlin&Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877),
with hmmalign
(HM_MER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm
(Thompson, J. D.,
Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80)
available e.g. on
http ://www.ebi . ac.uk/Tool s/clustalw/ or on http ://wvv-w. ebi
.ac.uk/Tools/clustalw2/index.html or
on http://npsa-pbil.ibcp.fr/cgi-bin/npsa automat.pl?page=/NPSA/npsa
clustalw.html. Preferred
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parameters used are the default parameters as they are set on
http://www.ebi.ac.uk/Tools/clustalw/
or http://www.ebi.ac.uk/Tools/c1usta1w2/index.html. The grade of sequence
identity (sequence
matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX).
BLAST protein
searches are performed with the BLASTP program, score = 50, word length = 3.
To obtain gapped
5 alignments for comparative purposes, Gapped BLAST is utilized as
described in Altschul et al.
(1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST
programs,
the default parameters of the respective programs are used. Sequence matching
analysis may be
supplemented by established homology mapping techniques like Shuffle-LAGAN
(Brudno M.,
Bioinformatics 2003b, 19 Supp11:154-162) or Markov random fields. Structure
based alignments
for multiple protein sequences and/or structures using information from
sequence database
searches, available homologs with 3D structures and user-defined constraints
may also be used
(Pei J, Grishin NV: PROMALS: towards accurate multiple sequence alignments of
distantly
related proteins. Bioinformatics 2007, 23:802-808; 3DCoffee@igs: a web server
for combining
sequences and structures into a multiple sequence alignment. Poirot 0, Suhre
K, Abergel C,
O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul 1;32:W37-40.). When
percentages of
sequence identity are referred to in the present application, these
percentages are calculated in
relation to the full length of the longer sequence, if not specifically
indicated otherwise.
Several documents are cited throughout the text of this specification. Each of
the documents
cited herein (including all patents, patent applications, scientific
publications, manufacturer's
specifications, instructions, etc.), whether supra or infra, are hereby
incorporated by reference in
their entirety. Nothing herein is to be construed as an admission that the
invention is not entitled
to antedate such disclosure by virtue of prior invention.
To practice the present invention, unless otherwise indicated, conventional
methods of
chemistry, biochemistry, and recombinant DNA techniques are employed which are
explained in
the literature in the field (cf. , e.g., Molecular Cloning: A Laboratory
Manual, 2nd Edition, J.
Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor
1989).
Throughout this specification and the claims which follow, unless the context
requires
otherwise, the word "comprise", and variations such as "comprises" and
"comprising", will be
understood to imply the inclusion of a stated integer or step or group of
integers or steps but not
the exclusion of any other integer or step or group of integers or steps. As
used in this specification
and the appended claims, the singular forms "a", "an", and -the" include
plural referents, unless
the content clearly dictates otherwise.
The term "TRBD" refers to an N-terminal polypeptide fragment of a ferritin
polypeptide of
between 15 to 40 amino acids, in particular approximately 20 amino acids, that
is capable of
specifically binding to CD71. Preferably, the TRBD has at least 50% of the
binding affinity to
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CD71 as the full length ferritin polypeptide, preferably at least 75%, more
preferably at least 90%.
It is well known in the art how to measure the binding affinity between two
proteins. Preferably,
the affinity between the TRBD and CD71, preferably between TRBD and CD71 of
the same
species, is measured by surface plasmon resonance at RT. Preferably, the KD of
the binding affinity
is 100 nM or lower, 50 nM or lower 20 nM or lower or 5 nM or lower.
In the following, the elements of the present invention will be described.
These elements are
listed with specific embodiments, however, it should be understood that they
may be combined in
any manner and in any number to create additional embodiments. The variously
described
examples and preferred embodiments should not be construed to limit the
present invention to only
the explicitly described embodiments. This description should be understood to
support and
encompass embodiments, which combine the explicitly described embodiments with
any number
of the disclosed and/or preferred elements. Furthermore, any permutations and
combinations of all
described elements in this application should be considered disclosed by the
description of the
present application unless the context indicates otherwise.
In a first aspect the present invention relates to a polypeptide comprising a
transferrin
receptor binding domain (TRBD) of a ferritin variant wherein within the TRBD
the ferritin variant
in comparison to the wild-type ferritin on which it is based comprises one or
more glutamine
residues (E) mutated into glutamic acid residues (Q) and/or one or more
asparagine residues (D)
mutated into aspartic acid residues (N). The ferritin variant, in particular
the TRBD of the ferritin
variant, comprises at least the following amino acid sequence:
MT TA SX1 SZ iVRZ2B YHZ 3DX2EAA ( SEQ ID NO. 81)
Xi = S or T, preferably T;
X2 = S or A, preferably S;
Zi, Z2, and Z3= Q or E; and
B = N or D;
wherein at least one of Z2 and Z3 is E and/or B is D. The amino acid sequence
may further
comprise one, two or three amino acid substitutions outside Z, in particular
outside Z2 and/or Z3,
and/or B. The M at position 1 may be present or absent. The amino acid
sequence according to
SEQ ID NO. 81 specifies the TRBD of the ferritin variant. In other words, the
polypeptide
according to the first aspect of the invention comprises a TRBD according to
SEQ ID NO. 81.It is
preferred that the polypeptide of the first aspect of the invention is a
ferritin polypeptide, i.e. a
polypeptide having a similar structure as a wild-type ferritin polypeptide
described below and/or
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an amino acid sequence that is homologous to the sequence of a wild-type
ferritin polypeptide
described below (e.g. sequence identity of at least 80%, 85%, 90% or 95% to a
wild-type ferritin
polypeptide described below). The polypeptide according to the first aspect of
the invention, i.e.
the polypeptide comprising a TRBD of a ferritin variant wherein within the
TRBD the ferritin
variant in comparison to the wild-type ferritin on which it is based comprises
one or more
glutamine residues mutated into glutamic acid residues and/or one or more
asparagine residues
mutated into aspartic acid residues, is also referred to as "TRBD variant
ferritin polypeptide" in
this specification.
The family of transferrin receptors comprises transferrin receptor 1 (TfR-1)
and transferrin
receptor 2 (TfR-2). TfR-1 is expressed on all actively proliferating cells,
while TfR-2 is expressed
mainly on hepatocytes and erythroid precursor cells. Transferrin receptor-1
has been shown to
mediate cellular uptake of ferritins via endocytosis. The designations "TfR-
1", "CD71", and
"TFRC" are used interchangeably and refer to transferrin receptor-1.
Ferritin is a hollow globular protein complex consisting of 24 ferritin
monomer subunits
assembled into a cage-like structure. Ferritin is the primary intracellular
iron-storage protein. It is
produced by almost all living organisms and is present in every cell type.
Ferritin genes are highly
conserved among species. In vertebrates, two ferritin monomers exist: the
light (L) chain and the
heavy (H) chain type with a molecular weight of 19 kDa or 21 kDa respectively.
Vertebrate ferritin
24-mers can be homo-oligomers consisting of either L or H chains, or hetero-
oligomers consisting
of both L and H chains (Theil EC, 1987, Annual Review of Biochemistry. 56 (1):
289-315):
Typically ferritin complexes have internal and external diameters of about 8
and 12 nm,
respectively. Ferritin was shown to be internalized by endocytosis upon
binding to CD71.
Interaction of ferritin and CD71 is mediated via ferritin-H chains (Li L et
al, Proc. Natl. Acad. Sci.
USA 107 (8) (2010) 3505-3510). Ferritins are not abundant in plasma, but can
be readily produced
in high yield as recombinant proteins in common protein expression systems
such as Escherichia
coli cells.
In the TRBD variant ferritin polypeptides according to the first aspect of the
invention,
uncharged amino acids (glutamine or asparagine) of the wild-type sequence are
substituted with
negatively charged amino acids (glutamic acid or aspartic acid). The inventors
surprisingly found
that this results in an increased affinity to CD71. Without wishing to be
bound by any theory, the
present inventors consider it likely that the negatively charged mutants
establish additional
interactions with the transferrin binding part of CD71, thus resulting in an
energetically more
favourable interaction between CD71 and the TRBD variant ferritin
polypeptides.
The TRBD variant ferritin polypeptides according to the first aspect of the
invention can be
described as "isosteric mutants", because compared to the respective wildtype
polypeptides, they
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exhibit an identical or very similar geometry. Compared to these "isosteric
mutants", mutants
carrying other mutations within the TRBD have a geometry that differs from
that of the wildtype.
Without wishing to be bound by any theory, the present inventors consider it
likely that the
resulting lack of surface complementarity with CD71 causes a reduced binding
affinity to CD71.
In a preferred embodiment of the polypeptide according to the present
invention, the wild-
type ferritin is a mammalian ferritin. The mammalian ferritin may be a mouse,
rat, dog, ape, in
particular chimpanzee, or human ferritin. In a preferred embodiment, the
mammalian ferritin is a
mouse, rabbit, rat or human ferritin.
In a preferred embodiment of the polypeptide according to the present
invention, the wild-
type ferritin is a human heavy chain ferritin.
In a preferred embodiment of the polypeptide according to the present
invention, the wild-
type ferritin has an amino acid sequence selected from the group consisting of
SEQ ID NO: 1,
which corresponds to the human ferritin heavy chain polypeptide and SEQ ID NO:
2, which
corresponds to the murine ferritin heavy chain polypeptide In a preferred
embodiment of the
polypeptide according to the present invention, the wild-type ferritin has an
amino acid sequence
according to SEQ ID NO: 1.
In a preferred embodiment of the polypeptide according to the present
invention, the
mutations are comprised in the 20 N-terminal amino acids of the wild-type
ferritin.
Amino acid substitutions are preferably selected in a way that they do not
unduly change the
conformation of the polypeptide, as a lack of surface complementarity with TfR-
1 will prevent
binding of the ferritin variant to CD71. As an example, a "small amino acid"
should be substituted
with another small amino acid. A "small amino acid" in the context of the
present invention is
preferably an amino acid having a molecular weight of less than 125 Dalton.
Preferably, a small
amino acid in the context of the present invention is selected from the group
consisting of the
amino acids glycine, alanine, serine, cysteine, threonine, and valine, or
derivatives thereof. As
another example, an amino acid having a hydrophobic side chain should be
substituted with
another amino acid having a hydrophobic side chain.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 05 to 18,
20 to 33, 35 to
48 and 50 to 63, which may further comprise one, two or three amino acid
substitutions outside
amino acid positions 11, 12 and/or 15.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO: 05 to SEQ
ID NO. 18,
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which may further comprise one, two or three amino acid substitutions outside
amino acid
positions 11, 12 and/or 15. SEQ ID NO. 04 to SEQ ID NO. 18 correspond to
single, double, triple
or quadruple mutants of the human ferritin TRBD sequence.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 05, 11,
12, 15, 20, 26, 27,
30, 35, 41, 42, 45, 50, 56, 57 and 60, which may further comprise one, two or
three amino acid
substitutions outside amino acid positions 11, 12 and/or 15.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 05, 11,
20, 26, 35, 41, 50
and 56, which may further comprise one, two or three amino acid substitutions
outside amino acid
position 11 These sequences correspond to mutants Q11E and 2ECSE (Q11E Q15E)
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 05, 20,
35 and 50, which
may further comprise one, two or three amino acid substitutions outside amino
acid position 11.
SEQ ID NO. 05, 20, 35 and 50 correspond to mutant Q11E.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 11, 26,
41 and 56, which
may further comprise one, two or three amino acid substitutions outside amino
acid positions 11
and 12. SEQ ID NO. 11, 26, 41 and 56 correspond to mutant EDCSE (Q11E N12D).
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 12, 27,
42 and 57, which
may further comprise one, two or three amino acid substitutions outside amino
acid positions 11
and 15. SEQ ID NO. 12, 27, 42 and 57 correspond to mutant 2ECSE (Q11E Q15E).
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least an
amino acid sequence selected from the group consisting of SEQ ID NO. 15, 30,
45 and 60, which
may further comprise one, two or three amino acid substitutions outside amino
acid positions 8,
11 and 15. SEQ ID NO. 15, 30,45 and 60 correspond to mutant 3ECSE (Q8E Q11E
Q15E).
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least SEQ
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ID NO. 05 (mutant Q11E), which may further comprise one, two or three amino
acid substitutions
outside amino acid position 11.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at SEQ ID
5 NO. 11, which may further comprise one, two or three amino acid
substitutions outside amino acid
positions 11 and 12.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least SEQ
ID NO. 12, which may further comprise one, two or three amino acid
substitutions outside amino
10 acid positions 11 and 15.
In a preferred embodiment of the polypeptide according to the first aspect of
the present
invention, the ferritin variant, in particular the TRBD of the ferritin
variant, comprises at least SEQ
ID NO. 15, which may further comprise one, two or three amino acid
substitutions outside amino
acid positions 8, 11 and 15
In a preferred embodiment of the polypeptide according to the present
invention, in addition
to the TRBD, the polypeptide further comprises an amino acid sequence having
at least 90%, 95%,
97%, 98%, 99% or 100%, such as 90%, 95%, 97%, 98%, 99% or 100%, identity to a
sequence
selected from the group consisting of SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID
NO. 78 to SEQ
ID NO. 80 and SEQ ID NO. 87. Preferably, this amino acid sequence is comprised
C-terminally
of the TRBD.
The polypeptide of the present invention essentially retains the properties of
a wild-type
ferritin polypeptide with regard to complex formation (cage-like structure
consisting of 24 ferritin
monomer subunits) and uptake of iron. The expression -essentially retains" is
meant to include
embodiments in which complex/24mer formations is improved compared to the wild-
type.
SEQ ID NO. 64 is an N-terminally truncated consensus sequence based on
sequences of
mammalian H-type ferritins. Within SEQ ID NO. 64 X at position 1 may be
present or absent, if
present it means any amino acid, preferably I, X at position 2 means any amino
acid, preferably
N, X at position 9 means any amino acid, preferably Y, X at position 19 means
any amino acid,
preferably C or Y, more preferable Y, X at position 61 means any amino acid,
preferably F, X at
position 63 means any amino acid, preferably Q, X at position 70 means any
amino acid, preferably
R or C, more preferably C, X at position 85 means any amino acid, preferably
H, X at position 89
means any amino acid, preferably S or N, more preferably N, X at position 116
may be present or
absent, if present it means any amino acid, preferably Y or H, more preferably
H, X at position
119 means any amino acid, preferably S or N, more preferably N, X at position
124 means any
amino acid, preferably S or A, more preferably A, X at position 143 means any
amino acid,
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11
preferably A or S, more preferably A, X at position 145 means any amino acid,
preferably M or
L, more preferably L, X at position 157 means any amino acid, preferably H or
D, more preferably
D, X at position 160 may be absent or any amino acid, preferably N, X at
position 161 may be any
amino acid, preferably E, X at position 162 may be any amino acid, preferably
S.
SEQ ID NO. 65 is an N-terminally and C-terminally truncated consensus sequence
based on
sequences of mammalian H-type ferritins. Within SEQ ID NO. 65 X at position 1
may be present
or absent, if present it means any amino acid, preferably 1, X at position 2
means any amino acid,
preferably N, X at position 9 means any amino acid, preferably Y, X at
position 19 means any
amino acid, preferably C or Y, more preferable Y, X at position 61 means any
amino acid,
preferably F, X at position 63 means any amino acid, preferably Q, X at
position 70 means any
amino acid, preferably R or C, more preferably C, X at position 85 means any
amino acid,
preferably II, X at position 89 means any amino acid, preferably S or N, more
preferably N, X at
position 116 may be present or absent, if present it means any amino acid,
preferably Y or H, more
preferably H, X at position 119 means any amino acid, preferably S or N, more
preferably N, X at
position 124 means any amino acid, preferably S or A, more preferably A.
SEQ ID NO. 66 is an alternative N-terminally truncated consensus sequence
based on
sequences of mammalian H-type ferritins. Within SEQ ID NO. 66 X at position 1
means any amino
acid, preferably N, X at position 8 means any amino acid, preferably Y, X at
position 60 means
any amino acid, preferably F, X at position 62 means any amino acid,
preferably Q, X at position
84 means any amino acid, preferably H, X at position 123 means any amino acid,
preferably S or
A, more preferably A, X at position 159 may be absent or any amino acid,
preferably N, X at
position 160 may be any amino acid, preferably E, X at position 161 may be any
amino acid,
preferably S.
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%,
98%, 99% or
100%, such as 90%, 95%, 97%, 98%, 99% or 100%, identity to a sequence selected
from the group
consisting of SEQ ID NO. 67 to SEQ ID NO. 70.
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises an amino acid sequence haying at least 90%, 95%, 97%,
98%, 99% or
100% identity to a sequence comprising amino acids 2-118 of SEQ ID NO. 67, SEQ
ID NO. 68,
SEQ ID NO. 69 or SEQ ID NO. 70.
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%,
98%, 99% or
100% identity to SEQ ID NO. 67. Within SEQ ID NO. 67, I at position 1 may be
present or absent.
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In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises an amino acid sequence having at least 90%, 95%, 97%,
98%, 99% or
100% identity to a sequence comprising amino acids 2-118 of SEQ ID NO. 67.
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises, essentially consists of or consists of a sequence
selected from the group
consisting of SEQ ID NO. 05 to SEQ ID NO. 63, which may further comprise one,
two or three
amino acid substitutions outside amino acid positions 11, 12 and/or 15; and a
sequence having at
least 90%, 95%, 97%, 98%, 99% or 100% identity to a sequence selected from the
group consisting
of SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ ID NO. 80 and SEQ ID
NO. 87 or
a sequence having 90%, 95%, 97%, 98%, 99% or 100% identity to a sequence
consisting of amino
acids 1-118 or 2-118 of SEQ ID NO. 64 to SEQ ID NO. 70, SEQ ID NO. 78 to SEQ
ID NO. 80,
or SEQ ID NO. 87, in particular SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69,
SEQ ID NO.
70, SEQ ID NO. 78 to SEQ ID NO. 80, or SEQ ID NO. 87, more particularly SEQ ID
NO. 78 to
SEQ ID NO 80, or SEQ TD NO 87, most particularly SEQ ID NO SO
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises, essentially consists of or consists of a sequence
selected from the group
consisting of SEQ ID NO: 05 to SEQ ID NO. 18 which may further comprise one,
two or three
amino acid substitutions outside amino acid positions 8, 11, 12 and/or 15 and
a sequence having
at least 90%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO. 67.
It is preferred that the polypeptide of the first aspect of the invention
comprises further
mutations compared to a wild-type ferritin sequence in the amino acid
sequences outside the
TRBD. In preferred embodiments, one, two, three or four, preferably four,
lysine residues,
preferably lysine residues at position 54, 72, 87 and/or 144 indicated with
respect to SEQ ID NO.
1 (human wild-type heavy chain ferritin), are deleted or substituted with a
non-basic amino acid.
A substitution with a non-basic amino acid is preferred over a deletion. A non-
basic amino acid
may be an acidic amino acid, such as D or E, an uncharged polar amino acid,
such as S, T, N or
Q, or an uncharged non-polar amino acid. Preferred is a substitution with E or
Q. Most preferably,
K54 is substituted with E, K72 is substituted with E, K87 is substituted with
Q and K144 is
substituted with E. The inventors surprisingly discovered that these mutations
enhance the
purification efficiency, decrease contamination, in particular contamination
with nucleic acids,
more particularly DNA, and endotoxins, increase the stability and improve the
encapsulation
efficiency (examples 3-8).
In some embodiments of the polypeptide of the first aspect of the invention,
one, two or
three, preferably two or three, cysteine residues, preferably cysteine
residues at position 91, 103
and/or 131 indicated with respect to SEQ ID NO. 1 (human wild-type heavy chain
ferritin), are
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13
deleted or substituted, preferably substituted with serine residues. The
inventors surprisingly
discovered that these mutations decrease the aggregation of ferritin
polypeptides into high
molecular weight complexes and improves the formation of 24-mers (examples 3-
4). In some
embodiments of the polypeptide of the first aspect of the invention one or
more lysine residues at
position 54, 72, 87 and/or 144 and one or more cysteine residues at position
91, 103 and/or 131
indicated with respect to SEQ ID NO. 1 are deleted or substituted as described
above.
In other embodiments, the cysteine residues are not mutated or deleted, as
they can be used
for covalent conjugation to drugs or labels, as claimed in the fifth aspect of
the invention.
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises, essentially consists of or consists of an amino acid
sequence selected from
the group consisting of SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ ID
NO. 74 SEQ
ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77 or an amino acid sequence having
at least 90%,
95%, 97%, 98%, or 99% identity to one of SEQ ID NO. 71-77. The polypeptide has
at least the
same affinity to TfR-1 and/or at least the same ability to form 24m ers as
wild-type human heavy
chain ferritin according to SEQ ID NO. 2.
In a preferred embodiment of the polypeptide according to the present
invention, the
polypeptide comprises, essentially consists of or consists of SEQ ID NO. 71.
In a preferred
embodiment of the polypeptide according to the present invention, the
polypeptide comprises,
essentially consists of or consists of SEQ ID NO. 72. In a preferred
embodiment of the polypeptide
according to the present invention, the polypeptide comprises, essentially
consists of or consists
of SEQ ID NO. 73. In a preferred embodiment of the polypeptide according to
the present
invention, the polypeptide comprises, essentially consists of or consists of
SEQ ID NO. 74.
In an even more preferred embodiment of the polypeptide according to the
present invention,
the polypeptide comprises, essentially consists of or consists of SEQ ID NO.
75, SEQ ID NO. 76,
or SEQ ID NO.77, most preferably SEQ ID NO. 77, or an amino acid sequence
having at least
90%, 95%, 97%, 98%, or 99% identity to one of SEQ ID NO. 75-77, preferably to
SEQ ID NO.
77.
In a preferred embodiment of the polypeptide according to the present
invention, the affinity
of the TRBD to TfR-1 is increased in comparison to the TRBD of the wild-type
ferritin to TfR-1
at least (>) 1.5x, > 2x, > 3x, > 4x, > 5x, > 10x, > 20x,? 30x, > 40x, > 50x,
but less than (<) 60x, <
50x, < 40x, < 30x, < 20x, < 10x, or < 5x. In this and the following
embodiments, "TRBD of the
wild-type ferritin" refers to the TRBD, in particular amino acids 1-20, of the
human or murine
ferritin heavy chain polypeptide according to SEQ ID NO. 1 or 2, respectively.
In a preferred
embodiment, the affinity of the TRBD to TfR-1 is increased at least 1.5x in
comparison to the
affinity of the TRBD of the wild-type ferritin to TR. In a preferred
embodiment, the affinity of the
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TRBD to TIER-1 is increased at least 2x in comparison to the affinity of the
TRBD of the wild-type
ferritin to TfR-1. In a preferred embodiment, the affinity of the TRBD to TfR-
1 is increased at
least 3x in comparison to the affinity of the TRBD of the wild-type ferritin
to TtR-1. In a preferred
embodiment, the affinity of the TRBD to TIER-1 is increased at least 4x in
comparison to the affinity
of the TRBD of the wild-type ferritin to TfR-1. In a preferred embodiment, the
affinity of the
TRBD to TIER-1 is increased at least 5x in comparison to the affinity of the
TRBD of the wild-type
ferritin to TfR-1. In a preferred embodiment, the affinity of the TRBD to TtR-
1 is increased at
least 10x in comparison to the affinity of the TRBD of the wild-type ferritin
to TfR-1. In a preferred
embodiment, the affinity of the TRBD to TIER-1 is increased at least 20x in
comparison to the
affinity of the TRBD of the wild-type ferritin to TfR-1. In a preferred
embodiment, the affinity of
the TRBD to TIER-1 is increased at least 30x in comparison to the affinity of
the TRBD of the wild-
type ferritin to TIER-i. In a preferred embodiment, the affinity of the TRBD
to TIER-1 is increased
at least 40x in comparison to the affinity of the TRBD of the wild-type
ferritin to TfR-1. In a
preferred embodiment, the affinity of the TRBD to TfR-1 is increased at least
50x in comparison
to the affinity of the TRBD of the wild-type ferritin to TIER-i. In a
preferred embodiment, the
affinity of the TRBD to TIER-1 is increased less than 60x in comparison to the
affinity of the TRBD
of the wild-type ferritin to TIER-i. In a preferred embodiment, the affinity
of the TRBD to TIER-1
is increased less than 50x in comparison to the affinity of the TRBD of the
wild-type ferritin to
TIER-i. In a preferred embodiment, the affinity of the TRBD to TfR-1 is
increased less than 40x in
comparison to the affinity of the TRBD of the wild-type ferritin to TIER-i. In
a preferred
embodiment, the affinity of the TRBD to TIER-1 is increased less than 30x in
comparison to the
affinity of the TRBD of the wild-type ferritin to TIER-i. In a preferred
embodiment, the affinity of
the TRBD to TIER-1 is increased less than 20x in comparison to the affinity of
the TRBD of the
wild-type ferritin to TIER-i. In a preferred embodiment, the affinity of the
TRBD to TIER-1 is
increased less than 10x in comparison to the affinity of the TRBD of the wild-
type ferritin to TIER-
1. In a preferred embodiment, the affinity of the TRBD to TfR-1 is increased
less than 5x in
comparison to the affinity of the TRBD of the wild-type ferritin to TIER-i. In
a preferred
embodiment, the affinity of the TRBD to TIER-1 is increased between 1.5x ¨ 50x
in comparison to
the TRBD of the wild-type ferritin. In a preferred embodiment, the affinity of
the TRBD to TIER-1
is increased between 2x ¨ 50x in comparison to the TRIM of the wild-type
ferritin. In a preferred
embodiment, the affinity of the TRBD to TIER-1 is increased between 3x ¨ 50x
in comparison to
the TRBD of the wild-type ferritin. In a preferred embodiment, the affinity of
the TRBD to TIER-1
is increased between 4x ¨ 50x in comparison to the TRBD of the wild-type
ferritin. In a preferred
embodiment, the affinity of the TRBD to TIER-1 is increased between 5x ¨ 50x
in comparison to
the TRBD of the wild-type ferritin. In a preferred embodiment, the affinity of
the TRBD to TIER-1
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is increased between 10x ¨ 50x in comparison to the TRBD of the wild-type
ferritin. In a preferred
embodiment, the affinity of the TRBD to TfR-1 is increased between 20x ¨ 50x
in comparison to
the TRBD of the wild-type ferritin. In a preferred embodiment, the affinity of
the TRBD to TfR-1
is increased between 30x ¨ 50x in comparison to the TRBD of the wild-type
ferritin. In a preferred
5 embodiment, the affinity of the TRBD to TfR-1 is increased between 40x ¨
50x in comparison to
the TRBD of the wild-type ferritin. In a preferred embodiment, the affinity of
the TRBD to TfR-1
is increased between 1.5x ¨ 10x in comparison to the TRBD of the wild-type
ferritin. In a preferred
embodiment, the affinity of the TRBD to TfR-1 is increased between 2x ¨ 20x in
comparison to
the TRBD of the wild-type ferritin. In a preferred embodiment, the affinity of
the TRBD to TfR-1
10 is increased between 5x ¨ 30x in comparison to the TRBD of the wild-type
ferritin.
An increased binding affinity of the TRBD to TfR-1 is advantageous because it
favors
binding of the TRBD variant ferritin polypeptides to TfR-1. This increases the
amount of TRBD
variant ferritin polypeptides bound to TfR-1 expressed on the surface of a
cell within a given time
and/or a given concentration of ferritin If an active ingredient is conjugated
to TRBD variant
15 ferritin polypeptides or encapsulated within oligomers of TRBD variant
ferritin polypeptides,
increased binding of TRBD variant ferritin polypeptides to TfR-1 facilitates
loading of a cell
expressing TfR-1 with the active ingredient.
In order to exert its function, an active ingredient conjugated to a TRBD
variant ferritin
polypeptide or encapsulated within an oligomer of TRBD variant ferritin
polypeptides has to be
released eventually. Without wishing to be bound by theory, the acidic pH of
the (late) endosomal
compartment may lead to disassembly of ferritin oligomers and thus to release
of the active
ingredient encapsulated within the oligomers. The present inventors have also
found that the
binding affinity of TRBD to TfR-1 should not be increased excessively, in
order not to completely
prevent dissociation of the TRBD variant ferritin polypeptide from TfR-1. In
order to be optimal,
the increase in affinity should not reach two orders of magnitude.
In a second aspect the present invention relates to a ferritin variant
polypeptide, wherein at
least one, at least two, at least three or at least four, preferably four,
lysine residues, preferably
lysine residues at position 54, 72, 87 and/or 144 indicated with respect to
SEQ ID NO. 1 (human
wild-type heavy chain ferritin), are deleted or substituted with a non-basic
amino acid. A
substitution with a non-basic amino acid is preferred over a deletion. A non-
basic amino acid may
be an acidic amino acid, such as D or E, an uncharged polar amino acid, such
as S, T, N or Q, or
an uncharged non-polar amino acid. The ferritin variant polypeptide is
characterized by a structural
and functional homology to a wild-type ferritin as defined above. The
inventors surprisingly
discovered that these mutations enhance the purification efficiency, decrease
contamination, in
particular contamination with nucleic acids, more particularly DNA, and
endotoxins, increase the
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16
stability and improve the encapsulation efficiency (examples 3-8). In some
embodiments, the
ferritin variant polypeptide of the second aspect comprises a mutation (i.e. a
deletion or
substitution) at position 54 and 72, or 54 and 87, or 54 and 144, or 72 and
87, or 72 and 144, or 87
and 144, or 54, 72 and 87, or 54, 72 and 144, or 54, 87 and 144, or 72, 87 and
144, or 54, 72, 87
and 144. It is preferred that the ferritin variant polypeptide comprises a
mutation at position 54,
72, 87 and/or 144. Most preferably, K54 is substituted with E, K72 is
substituted with E, K87 is
substituted with Q and K144 is substituted with E.
It is preferred that the ferritin variant polypeptide of the second aspect of
the invention has
a sequence according to SEQ ID NO. 82 (mammalian consensus), SEQ ID NO. 1
(human heavy
chain ferritin) or SEQ ID NO. 2 (murine heavy chain ferritin), wherein at
least one, preferably all,
lysine residues at position 54, 72, 87 and/or 144 are deleted or substituted
with a non-basic amino
acid, preferably E or Q, preferably the K at position 54 is substituted with
E, the K at position 72
is substituted with E, the K at position 87 is substituted with Q and/or the K
at position 144 is
substituted with E The sequences according to SEQ Ti) NO 82, SEQ TD NO 1 and
SEQ TD NO
2 may further comprise 1-5, e.g. 1,2, 3, 4 or 5, 1-10, e.g. 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10, 1-15, 1-20
or 1-25 amino acid mutations outside position 54, 72, 87 and/or 144.
In SEQ ID NO. 82, which is a mammalian consensus sequence, X at position 6 can
be any
naturally occurring amino acid, preferably Pro, X at position 14 can be any
naturally occurring
amino acid, preferably His, X at position 16 can be any naturally occurring
amino acid, preferably
Asp, X at position 21 may be present or absent, if present it means any amino
acid, preferably Ile,
X at position 22 means any amino acid, preferably Asn, X at position 30 can be
any naturally
occurring amino acid, preferably Tyr, X at position 40 can be any naturally
occurring amino acid,
preferably Tyr or Cys, more preferably Tyr, X at position 82 can be any
naturally occurring amino
acid, preferably Phe, X at position 84 can be any naturally occurring amino
acid, preferably Gln,
X at position 91 can be any naturally occurring amino acid, preferably Arg or
Cys, more preferably
Cys, X at position 106 can be any naturally occurring amino acid, preferably
His, X at position
110 can be any naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn, X at
position 137 can be any naturally occurring amino acid, preferably His or Tyr,
more preferably
His, X at position 140 can be any naturally occurring amino acid, preferably
Asn or Ser, more
preferably Asn, X at position 145 can be any naturally occurring amino acid,
preferably Ala or
Ser, more preferably Ala, X at position 164 can be any naturally occurring
amino acid, preferably
Ala or Ser, more preferably Ser, X at position 166 can be any naturally
occurring amino acid,
preferably Met or Leu, preferably Leu, X at position 178 can be any naturally
occurring amino
acid, preferably Asp or His, more preferably Asp, X at position 181 is absent
or any naturally
occurring amino acid, preferably Asn, X at position 182 is absent or any
naturally occurring amino
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17
acid, preferably Glu, X at position 183 is absent or any naturally occurring
amino acid, preferably
Ser.
Preferably, the ferritin variant polypeptide of the second aspect of the
invention has a
sequence according to SEQ ID NO. 82, SEQ ID NO. 1 or SEQ ID NO. 2 comprising
substitution
K54E or K72E or K87Q or K144E, or K54E and K72E, or K54E and K87Q, or K54E and
K144E,
or K72E and K87Q, or K72E and K144E, or K87Q and K144E, or K54E, K72E and
K87Q, or
K54E, K87Q and K144E, or K54E, K72E and K144E, or K72E, K87Q and K144E, or
K54E,
K72E, K87Q and K144E, preferably K54E, K72E, K87Q and K144E, wherein the
sequences
according to SEQ ID NO. 82, SEQ ID NO. 1 and SEQ ID NO. 2 may further comprise
1-5, e.g. 1,
2, 3, 4 or 5, 1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 1-15, 1-20 or 1-25
amino acid mutations outside
position 54, 72, 87 and/or 144
In preferred embodiments, the ferritin variant polypeptide of the second
aspect of the
invention further comprises isosteric mutations in the TRBD, in particular
mutation Q8E, Q1 1E,
N12D and/or Q15E, preferably Q11E or Q11E and Q15F, The i sosteric mutations
are indicated
with respect to the human wild-type ferritin sequence according to SEQ ID NO.
1 and are
described in the first aspect of the invention.
In some embodiments, in the ferritin variant polypeptide of the second aspect
of the
invention, one or more cysteine residues, in particular cysteine residues at
position 91, 103 and/or
131, are deleted or mutated, preferably mutated to serine residues.
In most preferred embodiments, the ferritin variant polypeptide of the second
aspect of the
invention has a sequence according to SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO.
85, SEQ ID
NO. 86, SEQ ID NO. 75, SEQ ID NO. 76, or SEQ ID NO. 77 or a sequence according
to SEQ ID
NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 75, SEQ ID NO.
76, or
SEQ ID NO. 77 comprising 1-5, e.g. 1,2, 3,4 or 5, or 1-10, e.g. 1, 2, 3, 4, 5,
6, 7, 8,9 or 10 amino
acid mutations outside position 54, 72, 87 and/or 144. In particularly
preferred embodiments, the
ferritin variant polypeptide of the second aspect of the invention has a
sequence according to SEQ
ID NO. 77.
SEQ ID NO. 86 is a mammalian consensus sequence, wherein each X has the same
meaning
as indicated above for SEQ ID NO: 82.
In a third aspect the present invention relates to a ferritin variant
polypeptide, wherein one
or more cysteine residues, in particular cysteine residues at position 91, 103
and/or 131 indicated
with respect to SEQ ID NO. 1, are deleted or substituted, preferably
substituted with serine
residues. The ferritin variant polypeptide is characterized by a structural
and functional homology
to a wild-type ferritin as defined above. The inventors surprisingly
discovered that these mutations
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decrease the aggregation of ferritin polypeptides into high molecular weight
complexes and
improves the formation of 24mers (examples 3-4).
It is preferred that the ferritin variant polypeptide of the third aspect of
the invention has a
sequence according to SEQ ID NO. 82, SEQ ID NO. 1 or SEQ ID NO. 2, wherein at
least one,
preferably all, cysteine residues at position 91, 103 and/or 131 are mutated,
preferably mutated to
serine residues. The sequences according to SEQ ID NO. 82, SEQ ID NO. 1 and
SEQ ID NO. 2
may further comprise 1-5, e.g. 1, 2, 3, 4 or 5, 1-10, e.g. 1,2, 3,4, 5, 6, 7,
8, 9 or 10, 1-15, 1-20 or
1-25 amino acid mutations outside position 91, 103 and/or 131.
In SEQ ID NO. 82, which is a mammalian consensus sequence, the X comprised in
the
sequence have the meaning outlined above.
In preferred embodiments, the ferritin variant polypeptide of the third aspect
of the invention
further comprises isosteric mutations in the TRBD, in particular mutation Q8E,
Q11E, N12D
and/or Q15E, preferably Q1 1E or Q1 1E and Q15E. The isosteric mutations are
indicated with
respect to the human wild-type ferritin sequence according to SEQ TD NO 1 and
are described in
the first aspect of the invention.
In preferred embodiments, the ferritin variant polypeptide of the third aspect
of the invention
further comprises a mutation at position 54, 72, 87 and/or 144. Preferably,
the mutations are
substitutions, in particular a substitution to E at position 54, a
substitution to E at position 72, a
substitution to Q at position 87 and/or a substitution to E at position 144.
These mutations are
further described in the second aspect of the invention.
In most preferred embodiments, the ferritin variant polypeptide of the third
aspect of the
invention has a sequence according to SEQ ID NO. 75 or SEQ ID NO. 76 or a
sequence according
to SEQ ID NO. 75 or SEQ ID NO. 76 comprising 1-5, e.g. 1, 2, 3, 4 or 5, or 1-
10, e.g. 1, 2, 3, 4,
5, 6, 7, 8, 9 or 10 amino acid mutations outside position 91, 103 and/or 131.
Active ingredients may be conjugated to the TRBD variant ferritin polypeptides
of the first
aspect of the invention or the polypeptides of the second or third aspect of
the invention or may
be encapsulated in oligomers of the TRBD variant ferritin polypeptides of the
first aspect of the
invention or the polypeptides of the second or third aspect of the invention.
The term "active
ingredient" encompasses therapeutically active ingredients and/or
diagnostically active
ingredients Thus, the term "active ingredient" refers to a therapeutic agent
(also referred to as
drug) and/or to a diagnostic agent (also referred to as label). The inventors
noted that the TRBD
variant ferritin polypeptides according to the invention represent preferred
constructs to
specifically deliver active ingredients, in particular encapsulated active
ingredients, to cells
expressing Tfit- 1. Furthermore, the inventors noted that ferritin variants
according to the invention
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19
are able to deliver active ingredients, in particular encapsulated active
ingredients, to the cell
nucleus.
The polypeptide according to the present invention may comprise additional
domains. In a
preferred embodiment of the polypeptide according to the present invention,
the polypeptide
further comprises an antigen binding domain, in particular an antibody or
antibody fragment.
The term "antibody" as used in the context of the present invention refers to
a glycoprotein
belonging to the immunoglobulin superfamily; the terms antibody and
immunoglobulin are often
used interchangeably. An antibody refers to a protein molecule produced by
plasma cells and is
used by the immune system to identify and neutralize foreign objects such as
bacteria and viruses.
The antibody recognizes a unique part of the foreign target, its antigen.
The term "antibody fragment" as used herein, refers to one or more fragments
of an antibody
that retain the ability to specifically bind to an antigen. Examples of
binding fragments
encompassed within the term "antibody fragment" include a fragment antigen
binding (Fab)
fragment, a Fab' fragment, a F(ab')/ fragment, a heavy chain antibody, a
single-domain antibody
(sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a
VH domain, a VL
domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new
antigen
receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-
targeting (DART)
molecule, a triple body, a diabody, a single-chain diabody, an alternative
scaffold protein, and a
fusion protein thereof.
The term "diabody- as used within this specification refers to a fusion
protein or a bivalent
antibody, which can bind different antigens. A diabody is composed of two
single protein chains,
which comprise fragments of an antibody, namely variable fragments. Diabodies
comprise a heavy
chain variable domain (VH) connected to a light-chain variable domain (VL) on
the same
polypeptide chain (VH-VL, or VL-VH). By using a short peptide connecting the
two variable
domains, the domains are forced to pair with the complementary domain of
another chain and thus,
create two antigen-binding sites. Diabodies can target the same (monospecific)
or different
antigens (bispecific).
A "single domain antibody", refers to antibody fragments consisting of a
single, monomeric
variable domain of an antibody. Simply, they only comprise the monomeric heavy
chain variable
regions of heavy chain antibodies produced by camelids or cartilaginous fish.
Due to their different
origins they are also referred to VHH or VNAR (variable new antigen receptor)-
fragments.
Alternatively, single-domain antibodies can be obtained by monomerization of
variable domains
of conventional mouse or human antibodies by the use of genetic engineering.
They show a
molecular mass of approximately 12-15 l(Da and thus, are the smallest antibody
fragments capable
of antigen recognition. Further examples include nanobodies or nanoantibodies.
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The term "antibody mimetic" as used within the context of the present
specification refers
to compounds, which can specifically bind antigens, similar to an antibody,
but are not structurally
related to antibodies. Usually, antibody mimetics are artificial peptides or
proteins with a molar
mass of about 3 to 20 kDa, which comprise one, two or more exposed domains
specifically binding
5
to an antigen. Examples include inter alia the LACI-Dl (lipoprotein-
associated coagulation
inhibitor); affilins, e.g. human-y B crystalline or human ubiquitin; cystatin;
Sac7D from
Sulfolobusacidocaldarius; lipocalin and anticalins derived from lipocalins;
DARPins (designed
ankyrin repeat domains); SH3 domain of Fyn; Kunitz domain of protease
inhibitors; monobodies,
e.g. the 10th type III domain of fibronectin; adnectins: knottins (cysteine
knot miniproteins);
10
atrimers; evibodies, e.g. CTLA4-based binders, affibodies, e.g. three-helix
bundle from Z-domain
of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin;
tetranectins, e.g.
monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-
inhibitor-II; affilins;
armadillo repeat proteins. Nucleic acids and small molecules are sometimes
considered antibody
mimetics as well (aptamers), but not artificial antibodies, antibody fragments
and fusion proteins
15
composed from these. Common advantages over antibodies are better
solubility, tissue
penetration, stability towards heat and enzymes, and comparatively low
production costs.
The term "antigen" is used to refer to a substance, preferably an immunogenic
peptide that
comprises at least one epitope, preferably an epitope that elicits a B or T
cell response or B cell
and T cell response.
20
An "epitope-, also known as antigenic determinant, is that part of a
substance, e.g. an
immunogenic polypeptide, which is recognized by the immune system. Preferably,
this
recognition is mediated by the binding of antibodies, B cells, or T cells to
the epitope in question.
In this context, the term -binding" preferably relates to a specific binding.
Epitopes usually consist
of chemically active surface groupings of molecules such as amino acids or
sugar side chains and
usually have specific three-dimensional structural characteristics, as well as
specific charge
characteristics. The term "epitope" comprises both conformational and non-
conformational
epitopes. Conformational and non-conformational epitopes are distinguished in
that the binding to
the former but not the latter is lost in the presence of denaturing solvents.
An immunogenic polypeptide according to the present invention is, preferably,
derived from
a pathogen selected from the group consisting of viruses, bacteria and
protozoa. However, in an
alternative embodiment of the present invention the immunogenic polypeptide is
a tumour antigen,
i.e. polypeptide or fragment of a polypeptide specifically expressed by a
cancer.
In a fourth aspect the present invention relates to a nucleic acid encoding
the polypeptide of
the first, second or third aspect.
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In another aspect the present invention relates to a vector comprising the
nucleic acid of the
fourth aspect.
In a fifth aspect the present invention relates to a conjugate comprising the
polypeptide of
the first, second or third aspect of the present invention and at least one
label and/or at least one
drug.
The phrase "conjugate comprising polypeptide and at least one label and/or at
least one drug"
as used in the context of the present invention refers to a composition in
which one or more
molecules of the active ingredient are covalently or non-covalently bound to a
polypeptide of the
first, second or third aspect of the invention. The covalent or non-covalent
binding between the
polypeptide and the active ingredient can be direct or indirect. In the latter
case the active
ingredient is linked to the polypeptide via a linker or spacer. Linker or
spacers are known to the
skilled artisan, such as polyalanine, polyglycine, carbohydrates, (CH2)11
groups or polypeptide
linkers, in particular peptide based cleavable linkers (e.g. cathepsin
sensitive valine-citnilline
sequence and para-aminobenzylcarbamate spacer). The skilled artisan will thus
be able to select
the respective suitable linker(s) or spacer(s) depending on the respective
application.
In a sixth aspect the present invention relates to a complex comprising at
least one
polypeptide of the first, second or third aspect of the present invention
and/or at least one conjugate
of the fifth aspect of the present invention.
The phrase "complex comprising polypeptide and/or at least one conjugate" as
used in the
context of the present invention refers to a complex formed by one or more
polypeptides of the
first, second or third aspect of the invention, by one or more conjugates of
the fifth aspect of the
invention, or by at least one polypeptide of the first, second or third aspect
of the invention and at
least one conjugate of the fifth aspect of the invention. The complex is
formed by covalent or non-
covalent binding between the polypeptide(s) and/or the conjugate(s). The
covalent or non-covalent
binding can be direct or indirect. In a preferred embodiment, the complex is
an oligomer, in
particular a 24-mer, formed by non-covalent binding between the polypeptide(s)
and/or the
conjugate(s).
In a preferred embodiment of the complex according to the invention, the
complex further
comprises at least one label and/or at least one drug.
The terms "drug" or "therapeutic agent" are used synonymously in the context
of the present
invention and refer to any compound that modifies or modulates cell activity
or is capable of being
activated, i.e. a prodrug, to modify or modulate cell activity, preferably in
the body of a patient.
Examples of such active ingredients include so called "small molecules" and
peptides. The term
"small molecule" is used in the context of the present invention to refer to a
hydrocarbon with a
molecular mass of below 1.500 g/mol or to pharmaceutically active radioactive
isotopes. Preferred,
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22
drugs that can be used comprise anti-cancer drugs, pharmaceutically active
radioactive isotopes or
ferrihydrite.
The term "prodrug" as used in the context of the present invention refers to
any active
ingredient that, after administration, is metabolized or otherwise converted
to a biologically active
or more active ingredient (or drug) with respect to at least one property. In
comparison to the drug,
a prodrug is modified chemically in a manner that makes it, relative to the
drug, less active or
inactive, but the chemical modification is such that the corresponding drug is
generated by
metabolic or other biological processes after the prodrug is administered to
the patient. A prodrug
may for example have, relative to the active drug, altered metabolic stability
or transport
characteristics, fewer side effects or lower toxicity, or improved flavor (for
example, see the
reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford
University
Press, New York, pages 388-392, incorporated herein by reference). A prodrug
may be synthesized
using reactants other than the corresponding drug.
The terms "label" or "diagnostic agent" are used interchangeably herein and
refer to any
kind of compound being suitable for diagnostic purposes. Preferred compounds
are selected from
a fluorescent dye, a radioisotope and a contrast agent. A contrast agent is a
dye or other substance
that helps to show abnormal areas inside the body. In one embodiment the term
label refers to a
compound that comprises a chelating agent which forms a complex with divalent
or trivalent metal
cations. Preferred radioisotopes/fluorescence emitting isotopes are selected
from the group
consisting of alpha radiation emitting isotopes, gamma radiation emitting
isotopes, Auger electron
emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as
65Tb, fluorescence
emitting isotopes, such as 15F, 51Cr, 67Ga, "Ga, "'In, 99mTc,140La, 175y10,
151 sm, 166H0,Y
RR,- 89Zr,
90y, 149pm, 177Lu, 47sc, 142pr, 159Grd, 212Bi, 72As, 72^e,
97RU, 109pd, 1051b, 101m11Rb, 119sb, 128Ba,
1231, 1241, 1311, 197Hg, 211At, 169Eu, 203pb, 212pb, 64cti,
iggRe, 186Re, 198Au and 199Ag as well as
conjugates and combinations of above with proteins, peptides, small molecular
inhibitors,
antibodies or other compounds, e.g. 18Ffluorodeoxyglucose (18F-FDG) or 64Cu-
porfirin. Preferred
fluorescent dyes are selected from the following classes of dyes: Xanthens
(e.g. Fluorescein),
Acridines (e.g. Acridine Yellow), Oxazines (e.g. Oxazine 1), Cynines (e.g. Cy7
/ Cy 3), Styryl
dyes (e.g. Dye-28), Coumarines (e.g. Alexa Fluor 350), Porphines (e.g.
Chlorophyll B), Metal-
Ligand-Complexes (e.g. PtOEPK), Fluorescent proteins (e.g. APC, R-
Phycoerythrin),
Nanocrystals (e.g. QuantumDot 705), Perylenes (e.g. Lumogen Red F300) and
Phtalocyanines
(e.g. IRDYETm700DX) as well as conjugates and combinations of these classes of
dyes or
fluorescent 65Tb emitting. Preferred contrast agents are selected from
paramagnetic agents, e.g.
Gd, Eu, W and Mn, preferably complexed with a chelating agent. Further options
are
superparamagnetic iron (Fe) complexes and particles, compounds containing
atoms of high atomic
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23
number, i.e. iodine for computer tomography (CT), microbubbles and carriers
such as liposomes
that contain these contrast agents.
The at least one label and/or at least one drug (i.e. at least one active
ingredient) is covalently
or non-covalently bound to a TRBD variant ferritin polypeptide according to
the first aspect of the
invention or a polypeptide according to the second or third aspect of the
invention or is
encapsulated within the complex according to the sixth aspect of the
invention. Thus, the term
-complex" also encompasses the enclosure of active ingredients within the cage
even in the
absence of a covalent or non-covalent bond between the protein(s) and the
active ingredient(s).
The formation of the complex allows the transport of the active ingredients
into the cell when the
cell is internalizing the ferritin. Thus, it is preferred that the active
ingredients are bound to the
iron binding protein in a way that does not interfere with the transport
mechanism. This can be
easily tested by the skilled person using uptake assays known in the art and
described in the
Example Section below. If the complex comprising an active ingredient is taken
up by a cell and
transported to a target region within the body, it is preferred that the
complex is sufficiently stable
to survive the transport within the cell to the target region within the body.
Thus, it is preferred
that the complex rather than the active ingredient alone is delivered to the
cells or into the cells in
the target region. This property also reduces possible deleterious effects,
e.g. cytotoxicity, of the
active ingredient to the cell delivering the active ingredient.
Active ingredients can be encapsulated within the internal cavity of a
ferritin oligomer
(physical confinement) by exploiting the association/dissociation properties
of the ferritin
macromolecule itself. The active ingredients are held in place by non-covalent
interactions with
amino acid residues within the cavity internal surface.
If active ingredients are covalently coupled to TRBD variant ferritin
polypeptides or
polypeptides according to the second or third aspect of the invention such
coupling is preferably
through amino acids residues known to be located in surface areas that are not
involved in binding
of ferritin to TfR-1. TRBD variant ferritin polypeptides used in the context
of the present invention
can form stable non-covalently bound complexes with a wide variety of active
ingredients. If the
active ingredient is a peptide, e.g. an antigenic peptide, it is preferred
that it is not expressed as a
fusion with the iron binding protein, since in this case release of the
peptide from the iron binding
protein will require endosomal processing of the entire ferritin peptide
fusion protein.
Whatever the conjugation/adsorption/binding method, the TRBD variant ferritin
polypeptides or polypeptides according to the second or third aspect of the
invention and the
described conjugates and complexes thereof were shown by the present inventors
to be privileged
carriers of drugs and labels, once loaded into appropriate cell systems with
tumour targeting
properties, e.g. activated macrophages. The purification procedure of these
TRBD variant ferritin
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24
polypeptides or polypeptides according to the second or third aspect of the
invention is easy, fast,
cheap and safe, which provides a tremendous added value.
The following preferred embodiments further specify both the fifth and sixth
aspect of the
present invention.
In a preferred embodiment, the drug and/or label is selected from the group
consisting of a
protein, a nucleic acid, a chemical non-protein non-nucleic acid compound with
a molecular
weight of less than 1.5 klla, more preferably less than 1 klla, a virus, and a
a- or 13-radiation
emitting radioisotope, which also emits a cell damaging amount of 7-radiation.
If the drug is a nucleic acid it is preferred that it is a miRNA, siRNA,
chemically modified-
RNA, LNA, ssRNA, DNAzyme or a nucleic acid encoding a pharmaceutically active
protein, e.g.
an antibody, an antibody mimetic, a cytokine, a prodrug-converting enzyme, an
immunogenic
peptide or the like
In a preferred embodiment, the label is selected from the group consisting of
a fluorescent
dye, a radioisotope/fluorescence emitting isotope, a detectable polypeptide or
nucleic acid
encoding a detectable polypeptide, and a contrast agent.
In a preferred embodiment, the fluorescent dye is selected from the group
consisting of the
following classes of fluorescent dyes: Xanthens, Acridines, Oxazines, Cynines,
Styryl dyes,
Coumarines, Porphines, Metal-Ligand-Complexes, Fluorescent proteins,
Nanocrystals, Perylenes
and Phtalocyanines as well as conjugates and combinations of these classes of
dyes.
In a preferred embodiment, the radioisotope/fluorescence emitting isotope is
selected from
the group consisting of alpha radiation emitting isotopes, gamma radiation
emitting isotopes,
Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent
isotopes, such as 65Tb,
fluorescence emitting isotopes, such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, 1111n,
99mTc, 140La,
175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As,
72Se, 97Ru,
109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 1231, 1241, 1311, 197Hg, 211At, 169Eu,
203Pb, 212Pb,
64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag as well as conjugates and
combinations of above
with proteins, peptides, small molecular inhibitors, antibodies or other
compounds (e.g. 18F-FDG,
89Zr-oxide or 64Cu-porfirin).
In a preferred embodiment, the detectable polypeptide is an autofluorescent
protein,
preferably green fluorescent protein or any structural variant thereof with an
altered adsorption
and/or emission spectrum.
In a preferred embodiment, the contrast agent comprises a paramagnetic agent,
preferably
selected from Gd, Eu, W and Mn, or ferrihydride.
In a preferred embodiment, the label comprises a chelating agent which forms a
complex
with divalent or trivalent metal cations.
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In a preferred embodiment of the targeted delivery system of the present
invention the
chelating agent is selected from the group consisting of 1,4,7,10-
tetraazacyclododecane-
N,N,N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA),
1,4,7-
triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA),
iminodiacetic acid,
5 D iethyl en etriam i ne-N,N,N',N',N"-pentaaceti c acid (D TPA) and 6-
Hydrazi nopyri di ne-3 -
carboxylic acid (HYNIC).
In a preferred embodiment, the drug is selected from the group consisting of
an anticancer
drug, an anti arteriosclerotic drug, and an anti-inflammatory drug or
immunomodulatory drug (e.g.
TRL agonists, STING agonists, mimicking viral or bacterial infection).
10 In a preferred embodiment, the anticancer drug is a cytostatic drug,
cytotoxic drug or prodrug
thereof.
Preferred anticancer drugs are selected from an apoptosi s/autophagy or
necrosis-inducing
drug. An apoptosis/autophagy or necrosis-inducing drug can be any drug that is
able to induce
apoptosi s/autophagy or necrosis effectively even in cells having an
abnormality in cell
15 proliferation. These drugs are preferably used in complexes with one or
more ferritins.
In a preferred embodiment, the anticancer drug is selected from the group
consisting of an
apoptosis-inducing drug, an alkylating substance, anti-metabolites,
antibiotics, epothilones,
nuclear receptor agonists and antagonists, an anti-androgene, an anti-
estrogen, a platinum
compound, a hormone, a antihormone, an interferon, an inhibitor of cell cycle-
dependent protein
20 kinases (CDKs), an inhibitor of cyclooxygenases and/or lipoxygenases, a
biogeneic fatty acid, a
biogenic fatty acid derivative, including prostanoids and leukotrienes, an
inhibitor of protein
kinases, an inhibitor of protein phosphatases, an inhibitor of lipid kinases,
a platinum coordination
complex, an ethyleneimine, a methylmelamine, a triazine, a vinca alkaloid, a
pyrimidine analog, a
purine analog, an alkylsulfonate, a folic acid analog, an anthracendione, a
substituted urea, and a
25 methylhydrazin derivative, an ene-diyne antibiotic, a tubulin
polymerization inhibitor such as a
maytansinoid or an auristatine derivate, immune check-point inhibitor, and an
inhibitor of tumour-
specific protein or marker, preferably a Rho-GDP-dissociation inhibitor, more
preferably Grp94,
or AXL inhibitor.
In a preferred embodiment, the anticancer drug is selected from the group
consisting of
acediasulfone, aclarubicine, ambazone, aminoglutethimide, auristatin, L-
asparaginase,
azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium
folinate, carboplatin,
carpecitabine, carmustine, celecoxib, chlorambucil, cis-platin, cladribine,
cyclophosphamide,
cytarabine, dacarbazine, dactinomycindapsone, daunorubicin, dibrompropamidine,
diethyl stilbestrole, docetaxel, doxorubicin, enediynes, epirubicin,
epothilone B, epothilone D,
estramucin phosphate, estrogen, ethinylestradi ole, etopo side, flavopiridol,
floxuridine,
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fludarabine, fluorouracil, fluoxymesterone, flutamidefosfestrol, furazolidone,
gemcitabine,
gonadotropin releasing hormone analog, hexamethylmelamine, hydroxycarbamide,
hydroxymethylnitrofurantoin, hydroxyprogesteronecaproat, hydroxyurea,
idarubicin, idoxuridine,
ifosfamide, interferon a, irinotecan, leuprolide, lomustine, lurtotecan,
mafenidesulfateolamide,
mechlorethamine, medroxyprogesterone acetate, megastrolacetate, melphalan,
mepacrine,
mercaptopurine, methotrexate, metronidazole, mitomycin C, mitopodozide,
mitotane,
mitoxantrone, mithramycin, nalidixic acid, nifuratel, nifuroxazide,
nifuralazine, nifurtimox,
nimustine, ninorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic
acid, pentamidine,
pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman,
prednimustine, prednisone,
preussin, procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib,
rosiglitazone,
sal azosul fapyri dine, scri fl avinium chloride,
semustine streptozoci ne, sul facarbami de,
sulfacetami de, sul fachlopyri dazi ne, sulfadi azi ne, sul fadi cram i de,
sulfadi m eth oxine, sul faethi dole,
sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole, sulfamethoxazole,
co-trimoxazole,
sul fam ethoxydi azin e, sul fam eth oxypyri dazi e,
sul fam oxol e, sulfanilamide, sul faperi n,
sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen, taxol,
teniposide,
tertiposide, testolactone, testosteronpropionate, thioguanine, thiotepa,
tinidazole, topotecan,
triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine,
vincristine, vindesine,
vinblastine, vinorelbine, and zorubicin, preferably selected from the group
consisting of auristatin,
banoxantrone, bendamustine, chlorambucil, chaliceamycin,
cyclophosphamidedynemycin A,
maytansine, melphalan, mertansine, and neocazinostatin, most preferably
banoxantrone,
bendamustine, chlorambucil, cyclophosphamide, pyrrolobenzodiazepine and
melphalan.
In a preferred embodiment, the anticancer drug is a proliferation inhibiting
protein,
preferably a cell cycle inhibitor or an antibody or antibody like binding
protein that specifically
binds to a proliferation promoting protein or a nucleic acid, preferably
encoding a proliferation
inhibiting protein or an antibody or antibody like binding protein that
specifically binds to a
proliferation promoting protein or a siRNA or DNAzyme.
In a preferred embodiment, the immunomodulatory drug activates or inhibits the
activity of
immune cells. These can be natural or synthetic ligands, including antibodies,
or antagonists of
Pattern Recognition Receptors, particularly Toll-like Receptors, NOD-like
receptors (NLR), RIG-
I-like receptors (RLR). Physiologically, these receptors recognize class of
signals known
as pathogen-associated molecular patterns (PAMPs) and damage-associated
molecular
patterns (DAMPs).
Preferred examples of antibodies to be used in the context of the present
invention are single
chain antibodies, antibody fragments, nanobodies, light or heavy chains,
variable light or variable
heavy chains, or diabodies. Preferred antibody fragments comprise a fragment
antigen binding
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27
(Fab) fragment, a Fab' fragment, a F(ab')2 fragment, a heavy chain antibody, a
single-domain
antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable
(Fv), a VH domain,
a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin
new antigen
receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-
targeting (DART)
molecule, a triple body, a diabody, a single-chain diabody, and a fusion
protein thereof
In a preferred embodiment, the virus is an oncolytic virus.
In a preferred embodiment, the a or 13 radiation emitting radioisotope, which
also emits a
cell damaging amount of 7 radiation is selected from the group consisting of
lutetium-177,
ytterbium-90, iodine-131, samarium-153, phosphorus-32, caesium-131, palladium-
103, radium-
233, iodine-125, and boron-10 or a cell damaging amount of a radiation,
preferably selected from
the group consisting of actinium-225, bismuth-213, lead-212, and polonium-212.
Also preferred
is a complex of above mentioned compounds and isotopes linked to the
nanoparticles (e.g. gold,
argentum, graphen) or these nanoparticles.
In a preferred embodiment, the drug is a hypoxi a-activated prc-)drug,
preferably selected from
the group consisting of benzotriazine N-oxides, apaziquone (E09), tirapazamine
(TPN), SN30000,
PR-104A, TH-302, TH-4000, AQ4N.
In a preferred embodiment, the drug is an antigen or a nucleic acid encoding
an antigen.
In a preferred embodiment, the bond(s) between the TRBD variant ferritin
polypeptide or
polypeptides according to the second or third aspect of the invention and the
active ingredient in
the conjugate are covalent and/or non-covalent; and/or the active ingredient
comprised in the
complex is entrapped/encapsulated by the oligomers of the TRBD variant
ferritin polypeptide. In
one embodiment the covalent and/or non-covalent coupling is indirect through a
linker or spacer.
If the formation of covalent bonds is desired, relevant thiol, amino or
carboxyl groups of the TRBD
variant ferritin polypeptides are used to covalently couple active ingredients
modified by specific
active linker moieties reactive towards thiol or amino groups directly or
indirectly to the TRBD
variant ferritin polypeptides.
TRBD variant ferritin polypeptides or polypeptides according to the second or
third aspect
of the invention may be linked to cysteine thiol reactive active ingredients
bearing a peptide based
cleavable linker (e.g. cathepsin sensitive valine-citrulline sequence and para-
aminobenzylcarbamate spacer). As a notable example, the antimitotic agent
monomethylauristatin
E (MMAE) has been used. The peptide-based linker binds the protein to the
cytotoxic compound
in a stable manner so the drug is not easily released from the protein under
physiologic conditions
and help prevent toxicity to healthy cells and ensure dosage efficiency. The
ferritin active
ingredient adduct thus generated is capable of attaching to the selected
receptor type, i.e. TIR-1
for ferritin. Once bound the ferritin active ingredient adduct is internalised
by endocytosis and thus
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28
selectively taken up by targeted cells. The vesicle containing the drug is
fused with lysosomes and
lysosomal cysteine proteases, particularly cathepsin B start to break down the
valine-citrulline
linker and MMAE is no longer bound to ferritin and is released directly into
the tumour
environment.
Alternatively, DM1-SMCC is an efficient mertansine derivative bearing a linker
that
specifically binds to lysine residues generating a covalent complex with
ferritin, in a reaction that
has been successfully described for antibodies. In particular, ferritin can be
reacted with DM1-
SMCC thus providing a covalent protein-drug adduct that can be cleaved inside
cells and releases
the active drug in a time-dependent manner. The suppression of microtubule
dynamics by DM1
induces mitotic arrest and cell death.
The term "full load" is used in the context of the present invention to refer
to the maximum
amount of ferritin complexed with an active ingredient that can be taken up by
the cell of the active
delivery system.
Tt is also envisioned that different pharmaceutically active substances,
labels or
pharmaceutically active substances and labels are comprised in the complex
according to the third
aspect of the invention. For example, one type of active ingredient may be
bound to a TRBD
variant ferritin polypeptide or polypeptides according to the second or third
aspect of the invention
(non-covalently bound), while another type is encapsulated in the complex.
This approach utilizes
different release rates of the active ingredients from the complex once
delivered to the targeted
tissue and/or cells. For example, an active ingredient can be covalently
attached to a ferritin
molecule either on the surface of the 24-mer or within the internal cavity by
exploiting the
reactivity of relevant thiol, amino or carboxyl groups. The types of such
useful reactions are well
known in the art and can be adopted by the person skilled in the art to the
particular active
ingredient without any additional work. Examples of such reactions are
described in Behrens CR,
Liu B. Methods for site-specific drug conjugation to antibodies. MAbs. 2014
Jan-Feb;6(1):46-53.
In theragnostic applications, i.e. in which the complex comprises both a label
and a drug,
it is preferred that the label is covalently attached to the iron binding
protein and the drug is non-
covalently bound to the iron binding protein and/or entrapped in the internal
cavity formed upon
assembly of the multimer of TRBD variant ferritin polypeptides or polypeptides
according to the
second or third aspect of the invention.
In a sixth aspect, the present invention relates to an isolated targeted
delivery system
comprising a cell, wherein the cell comprises the polypeptide of the first,
second or third aspect,
the conjugate of the fifth aspect or the complex of the sixth aspect of the
present invention.
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29
The term "targeted delivery system" refers to a system that is capable of
delivering an active
ingredient to the targeted region, i.e. of capable of targeted delivery,
preferably within the body of
a patient.
The term "targeted delivery" refers to the delivery of a therapeutic or
diagnostic agent
(herein together referred to also as "active ingredient") to a subject, e.g.
patient, in particular to a
cell within the body of a patient. Targeted delivery also includes "targeted
theragnostic delivery",
meaning that both a therapeutic and a diagnostic agent are delivered
concomitantly, preferably to
a diseased region, thus allowing simultaneous treatment and diagnosis and/or
treatment
monitoring.
Targeted delivery results in an increased concentration of the active
ingredient in a particular
region of the body when compared to other regions of the body of that patient.
Preferably, the
relative concentrations are compared between a diseased region(s) of the body
and other regions
of the body having similar access to the blood circulation. In preferred
embodiments the
concentration of the active ingredient in a given number of cells or a given
biopsy volume from
the diseased region is at least 10% higher, if compared to the identical
number of cells or biopsy
volume from a non-diseased region after administration of the targeted
delivery system of the
present invention, preferably after 2-24 hrs. More preferably, the
concentration of the active
ingredient in the diseased region of the body of a patient is at least 20%, at
least 30%, at least 40%,
at least 50%, at least 60%, at least 70% at least 80%, at least 90%, at least
100%, at least 150%, at
least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at
least 450%, at least
500%, more preferably at least 1000% higher than in a non-diseased region of
the body after
administration of the targeted delivery system of the present invention,
preferably after 2-24 hrs.
When assessed on the basis of total body distribution it is preferred that at
least 5% of the active
ingredient administered to a patient is delivered to the diseased region of
the body, preferably at
least 10%, more preferably at least 15%. The targeted delivery of the active
ingredient limits the
potential deleterious effects of an active ingredient to the diseased region
of the body.
The targeted delivery system according to the present invention enables tumour
delivery of
the pharmaceutically active substances, labels or pharmaceutically active
substances and labels,
which normally would not be able to reach the tumour (for example, due to
solubility problems).
This allows precise administration of the active ingredients to the tumour
site (especially to the
hypoxic regions) and into the tumour mass, avoiding their accumulation in
other organs.
Targeted delivery encompasses both direct and indirect targeting. Direct
targeting refers to
direct uptake of an active ingredient (as a conjugate according to the second
aspect of the invention
or in a complex according to the third aspect of the invention) by a diseased
cell, e.g. a cancer cell.
Indirect targeting refers to delivery of an active ingredient (as a conjugate
according to the second
CA 03212386 2023- 9- 15
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aspect of the invention or in a complex according to the third aspect of the
invention) to a diseased
cell, e.g. a cancer cell, by another cell, e.g. a leukocyte cell.
Mutant Q1 lE was shown to be capable of at least fourfold higher binding
affinity to the
CD71 receptor and a corresponding slow rate of release from its complex. The
mutant is thus
5 readily taken up by cancer cells overexpressing CD71 receptors (direct
targeting). In addition, the
mutant is readily taken up by CD45+ leukocytes capable of transferring the
ferritin mutants to
target cells (indirect targeting).
In particular if the cells of the targeted delivery system of the present
invention are
leukocytes, the targeted delivery system targets lymph nodes, which makes it
particularly suitable
10 for delivery of antigens to dendritic cells residing in the lymph nodes.
The lymph node targeting
is particularly pronounced, if the cells loaded with the complex are
macrophages in particular
activated macrophages, even more preferably CCL-2 activated bone marrow
derived activated
macrophages, or lymphocytes, in particular B cells or T cells. Thus in a
preferred embodiment the
targeted delivery system is used to deliver one or more antigens in order to
elicit a prophylactic
15 and/or therapeutic immune response against the one or more antigens.
Preferred antigens are
derived from pathogens, i.e. bacteria or viruses or are tumour specific
antigens. The term "tumour
specific antigens" refers to proteins or epitopes (including peptides with
altered glycosylation
patterns) that are higher expressed on tumour cells in comparison to non-
tumour cells, preferably
to antigens or epitopes only expressed on tumour cells. Preferred antigens are
selected from the
20 group consisting of epidermal growth factor receptor (EGFR, ErbB-1, HERO,
ErbB-2
(HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-like growth
factor
receptor (IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligand family;
platelet derived
growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth
factor receptor
(FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor
(VEGFR)
25 family, VEGF family; HGF receptor family; TRK receptor family; ephrin
(EPH) receptor family;
AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE
receptor family,
angiopoietin 1,2; receptor tyrosine kinase-like orphan receptor (ROR) receptor
family; discoidin
domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK
receptor family;
MuSK receptor family; Transforming growth factor a (TGF-a) receptors, TGF-P;
Cytokine
30 receptors, Class I (hematopoietin family) and Class II (interferon/IL-10
family) receptors, tumor
necrosis factor (TNF) receptor superfamily (TNFRSF), death receptor family;
cancer-testis (CT)
antigens, lineage-specific antigens, differentiation antigens, alpha-actinin-
4, ARTC1, breakpoint
cluster region-Abelson (Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5),
caspase-8 (CASP-
8), 13-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent
kinase 4 (CDK4),
CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2),
Ets variant
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31
gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein,
fibronectin (FN),
GPNMB, low density lipid receptor/GDP-L fucose: 13-Dgalactose 2-a-
Lfucosyltransferase
(LDLR/FUT) fusion protein, HLA-A2. arginine to isoleucine exchange at residue
170 of the cc-
helix of the 02-domain in the HLA-A2 gene (HLA-A*201-R170I), HLA-A11, heat
shock protein
70-2 mutated (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutated 1, 2, 3
(MUM-1,
2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin class I, NFYC, OGT,
OS-9, pml-
RARalpha fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS,
RBAF600,
SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triosephosphate isomerase,
BAGE,
BAGE-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetyl
glucosaminyl
transferase V. MGAT5), HERV-K-MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-
recognized
antigen on melanoma (CAMEL), MACE-Al (MACE-1), MAGE-A2, MAGE-A3, MAGE-A4,
MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A 1 1, MAGE-Al2,
MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, mucin 1
(MUC1), MART-1/1Vfelan-A (MI,ANA), gpl 00, gp100/Pmel 1 7 (STT,V), tyrosinase
(TYR), TRP-
1, HAGE, NA-88, NY-ESO-1, NY-ES0-1/LAGE-2, SAGE, Sp17, SSX-1,2,3,4, TRP2-INT2,
carcino-embryonic antigen (CEA), kallikrein 4, mammaglobin-A, A1, prostate
specific antigen
(PSA), TRP-1/gp75, TRP-2, adipophilin, interferon inducible protein absent in
melanoma 2 (AIM-
2), BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM),
EphA3, fibroblast
growth factor-5 (FGF-5), glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu
(ERBB2),
interleukin 13 receptor ct2 chain (IL13Ralpha2), IL-6 receptor, intestinal
carboxyl esterase (ICE),
alpha-feto protein (AFP), M-C SF, mdm-2, MUC1, p53 (TP53), PBF, PRAME, PSMA,
RAGE-1,
RNF43, RU2AS, SOX10, STEAP1, survivin (BIRC5), human telomerase reverse
transcriptase
(hTERT), telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2,
PAGE-5, LIP1, CTAGE-1, C SAGE, M1VIA1, CAGE, BORIS, HOM-TES-85, AF15q14,
HCA661,
LDHC, MORC, SGY-1, SP011, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE,
TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors (ER),
androgen
receptors (AR), CD40, CD30, CD20, CD19, CD33, cancer antigen 72-4 (CA 72-4),
cancer antigen
15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125),
cancer antigen
19-9 (CA 19-9), 13-human chorionic gonadotropin, 1-2 microglobulin, squamous
cell carcinoma
antigen, neuron-specific enolase, heat shock protein gp96, GM2, sargramostim,
CTLA-4, 707
alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-
4),
carcinoembryogenic antigen peptide-1 (CAP-1), calcium-activated chloride
channel-2 (CLCA2),
cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), Human papilloma
virus (HPV)
proteins (HPV-E6, HPV-E7, major or minor capsid antigens, others), Epstein-
Barr virus (EBV)
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32
proteins (EBV latent membrane proteins - LMP1, LMP2; others), Hepatitis B or C
virus proteins,
and HIV proteins.
The targeted delivery system of the present invention has particular
suitability to deliver
active ingredients to hypoxic areas. Hypoxia is characteristic of various
disease including cancer
and inflammatory diseases and thus allows targeting such diseases.
In addition to the targeting the use of active ingredients, which are
activated under hypoxic
conditions adds a further specificity to the targeting and/or further reduces
adverse effects of the
active ingredients. Thus, in particularly preferred embodiments the active
ingredient is a hypoxia-
activated prodrug. The backbone of all the hypoxia-activated prodrugs is the
presence of one of
five different chemical moieties (nitro groups, quinines, aromatic and
aliphatic N-oxides and
transition metals) that are enzymatically reduced under hypoxic conditions in
tissue. Hypoxia¨
activated prodrugs are any prodrug that is less active or inactive, relative
to the corresponding
drug, and comprises the drug and one or more bioreducible groups. Such
hypoxia¨activated
prodrugs include all prodnigs activated by a variety of reducing agents and
reducing enzymes,
including without limitation single electron transferring enzymes (such as
cytochrome P450
reductases) and two electron transferring (or hydride transferring) enzymes.
According to
preferred embodiment of the invention hypoxia¨activated prodrug is TH-302.
Methods of
synthesizing TH-302 are described in PCT application WO 07/002931 and WO
08/083101.
Preferably examples of such prodrugs are selected from the class I group
consisting of:
benzotriazine N-oxides, apaziquone (E09), tirapazamine (TPN) and SN30000; or
class II group
consisting of: nitro compounds PR-104A, TH-302, TH-4000, and AQ4N.
A striking observation was the disease specific homing of the targeted
delivery system of
the present invention. In particular the CD45+ leukocyte cells appear to have
a tropism for hypoxic
areas and areas of oxidative stress. Hypoxia is a hallmark of various diseases
as is oxidative stress.
Accordingly, the present invention also relates to an isolated targeted
delivery system of the fourth
aspect of the invention for use in preventing, treating or diagnosing a
disease characterized by
hypoxic areas within the diseased tissue and/or by areas of oxidative stress,
in particular hypoxic
tumours or a hypoxic area within a tumour, or any area within an organism
subjected to hypoxic
conditions, for example during ischaemic incidents, or undergoing an
inflammatory process.
Similarly, the invention relates to a method of treating, preventing or
diagnosing a disease
characterized by hypoxic areas within the diseased tissue and/or by areas of
oxidative stress, in
particular hypoxic tumours or a hypoxic area within a tumour, or any area
within an organism
subjected to hypoxic conditions, for example during ischaemic incidents, or
undergoing an
inflammatory process, by administering an effective amount of the isolated
targeted delivery
system of the fourth aspect of the invention to a subject in need thereof
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33
The ability of a given cell or of a population thereof to internalize ferritin
depends on the
expression of receptors involved in this internalization process. Receptors
that lead to
internalization of ferritin comprise, e.g. TfR, CXCR4, scavenger receptors,
CD163, and TIM-2.
The skilled person is well aware how to measure the amount of ferritin uptake
and preferred
methods of measuring the uptake are described in the Example Section below.
In a preferred embodiment of the isolated targeted delivery system according
to the
invention, the cell is a CD45+ leukocyte, in particular a CD45 leukocyte
selected from the group
consisting of a monocyte, a differentiated monocyte, a monocyte-macrophage, a
lymphocyte and
a granulocyte.
The term -leukocyte" (or "leukocyte cell") is used in the context of the
present invention to
refer to cells of the immune system that are involved in protecting the body
against both infectious
disease and foreign invaders. All leukocytes are produced and derived from
multipotent cells in
the bone marrow known as a hematopoietic stem cells. Leukocytes are found
throughout the body,
including the blood and lymphatic system All leukocytes have nuclei, which
distinguishes them
from the other blood cells, the anucleated red blood cells (RBCs) and
platelets. Types of leukocyte
can be classified in standard ways. Two pairs of the broadest categories
classify them either by
structure (granulocytes or agranulocytes) or by cell division lineage (myeloid
cells or lymphoid
cells). These broadest categories can be further divided into the five main
types: neutrophils,
eosinophils, basophils, lymphocytes, and monocytes. These types are
distinguished by their
physical and functional characteristics. Monocytes and neutrophils are
phagocytic. Further
subtypes can be classified; for example, among lymphocytes, there are B cells,
T cells, and NK
cells. Granulocytes are distinguished from agranulocytes by their nucleus
shape (lobed versus
round, that is, polymorphonuclear versus mononuclear) and by their cytoplasm
granules (present
or absent, or more precisely, visible on light microscopy or not thus
visible). The other dichotomy
is by lineage: Myeloid cells (neutrophils, monocytes, eosinophils and
basophils) are distinguished
from lymphoid cells (lymphocytes) by hematopoietic lineage (cellular
differentiation lineage).
CD45+ expression is characteristic of a subgroup of leukocyte cells, i.e.
monocyte,
monocyte-macrophages, lymphocytes, granulocytes, NK cells that are suitable to
be used in the
context of the targeted delivery system of the present invention, in
particular since CD45+
leukocyte cells are attracted to particular tissues and cells within the body
and are capable of
delivering complexes of one or more iron binding proteins and one or more
pharmaceutically
active substances, labels or pharmaceutically active substances and labels to
or into cells. This
subgroup of leukocytes is in the following referred to as "CD45+ leukocyte
cells" or "CD45+
leukocytes". Preferably the monocyte is not a dendritic cell which
differentiation is controlled by
one or more of the following transcription factors: IFN-regulatory factor 8
(IRF8), nuclear factor
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34
interleukin (IL)-3-regulated protein (NFIL3), basic leucine zipper
transcriptional factor ATF-like
3 (BATF3) or Transcription Factor RelB (NF-KB Subunit) - RELB, Spi-1 Proto-
Oncogene (PU/1), recombining binding protein suppressor of hairless (RBPJ),
IFN-regulatory
factor 4 (IRF4) or transcription factor E2-2 (also known as (TCF4).
It is understood by the skilled person that CD45+ leukocyte cells as defined
above unless of
clonal origin are a mixed population of different leukocytes which share the
common property of
expressing CD45 surface antigen. Accordingly, subpopulations of cells within
the diverse group
of CD45+ leukocyte cells as defined above are characterized throughout the
specification by
further functional and/or structural characteristics. The term -CD45+"
indicates that the majority
of cells within a population of cells or essentially all cells express the
CD45+ surface antigen.
"Expressing" means in this respect that the majority of cells within a
population of cells or
essentially all cells express the marker (also called surface antigen herein)
In this context and also
with reference to other cellular surface antigens, the term "expresses"
indicates that the surface
antigen is produced within the cell and detectably exposed on the surface of a
cell The level of
expression and, thus the number of surface antigens detectably exposed on the
surface of a cell
can vary greatly among different cells. Generally, a cell is considered to be
positive, i.e. is
indicated to be "k", for a cellular surface antigen, if at least 5, preferably
at least 10 copies of the
surface antigen are detectably exposed on the surface of the cell. The skilled
person is well aware
of how to detect, quantify and select for cells, which are positive (or
negative) for a given cellular
surface antigen. Preferred methods include Fluorescence Activated Cell Sorting
(FACS). In this
technology fluorescently labelled antibodies are used to bind to cellular
surface antigens of a
population of cells, the cells are subsequently isolated into single cells and
based on fluorescence
intensity measured for the single cell, characterized as being positive or
negative for the given
cellular surface antigen. In some embodiments of the present invention it is
indicated that the
expression of a given protein is high or low. This means that the protein is
detectably expressed in
both instances, i.e. is "k", however, at different levels. High and low
expression, respectively, will
mean different absolute numbers of proteins per cell for different proteins.
Thus, a given protein
may be considered to be expressed at high levels if there are more than 500
detectable copies of
that protein per cell and to be expressed at low levels if there are between
Ito 50 detectable copies
of that protein per cell. However, another protein may be considered to be
expressed at high levels,
if there are more than 5000 detectable copies and expressed at low levels, if
there are between 1
to 500 detectable copies per cell. It is well known in the art how to quantify
the number of proteins
expressed or produced in a cell using flow cytometry and Becton Dickinson
QuantibriteTM bead
method (see e.g. Pannu, K.K., 2001, Cytometry. 2001 Dec 1;45(4):250-8) or mass
spectrometry
(see, e.g. Milo, R., 2013, Bioessays, 35(12): 1050-1055).
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For the purpose of the present invention the term "high expression" of a given
protein refers
to detectable expression of that protein that is at least 70% of the highest
expression level found,
i.e. number of copies per cell, in a population of healthy cells, in
particular CD45-' leukocytes. The
term "low expression" of a given protein refers to detectable expression of
that protein that is 30%
5 or less of the highest expression level found, i.e. number of copies of
that protein per cell, in a
population of healthy cells, in particular CD45-P leukocytes. Preferably, the
"highest expression
level" is determined as the average of the highest expression levels found in
healthy cells, in
particular CD45- leukocytes of different subjects. In some embodiments
preferred subpopulations
of cells are characterized as "producing" a given protein. This is understood
to mean that the
10 protein is not necessarily detectable on the surface of the cell but may
only be present inside the
cell. The skilled person is well aware how to detect and/or quantify
production of a protein inside
a cell and/or select cells producing such proteins. Alternatively, cell
populations can be defined by
expression of specific transcription factors It is well known in the art how
to determine expression
of a given protein or its encoding mRNA in a population of cells or even in
single cells, e.g. using
15 in vivo labelling with antibodies, FISH assays, in vivo single molecule
fluorescent microscopy
(Crawford, R. et al. Biophys J. (2013) 105(11): 2439) alone or in combination
with Fluorescent
Activated Cell Sorting (FACS), or by the PrimeFlow technique (e Bioscience),
(Adam S.
Venable, el. al., (2015) Methods in Molecular Biology).
The term "differentiated monocyte" is used in the context of the present
invention to refer
20 to a monocyte differentiated from the committed precursor termed macrophage-
DC precursor
(MDP) mainly resident in bone marrow (but could be also in the spleen) and
differentiate into
either dendritic cells or macrophages. In mice they consist of two main
subpopulations: (i) CD11b
cell with high expression of CX3CR1, low expression of CCR2 and Ly6C- and (ii)
CD1 lb" cell
with low expression of CX3CR1, high expression of CCR2 andLy6C. After leaving
the bone
25 marrow, mouse Ly6C-P monocytes differentiate into Ly6C- monocytes in
circulation. Similarly, in
human monocyte differentiation, it is accepted that CD14-' classical monocytes
leave bone
marrow and differentiate into CD14'CD16+ intermediate monocytes and
sequentially to
CD14-PCD16-'non-classical monocytes in peripheral blood circulation (Yang et
al. 2014; Biomark
Res 2(1) doi. 10.1186/2050-7771-2-1). Preferably the differentiated monocyte
is not a dendritic
30 cell, which differentiation is controlled by one or more of the
following transcription factors: IRF8,
NFIL3, BATF3, RELB, PU/1, RBPJ, IIRF4, and/or TCF4, and more preferably is not
a dendritic
cell.
Macrophages are tissue-resident professional phagocytes and antigen-presenting
cells
(APC), which differentiate from circulating peripheral blood monocytes (PBMs).
The term
35 "activated macrophage" is used in the context of the present invention
to refer to any macrophage
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36
that is polarized. Macrophage activation is in general achieved by incubation
with interleukins,
cytokines and/or growth factors. In particular IL-4 and M-CSF can be used as
activating agents.
Activated macrophages of different phenotypes are classified into M1 -
macrophages, classically
activated macrophages (CAM) and M2-macrophages, alternatively activated
macrophages
(AAIV1). The classically activated Ml-macrophages comprise immune effector
cells with an acute
inflammatory phenotype. These are highly aggressive against bacteria and
produce large amounts
of lymphokines (Murray, and Wynn, 2011, .1 Leukocl3iol, 89(4):557-63). The
alternatively
activated, anti-inflammatory M2-macrophages can be separated into at least
three subgroups.
These subtypes have various different functions, including regulation of
immunity, maintenance
of tolerance and tissue repair/wound healing. The term "Ml inducer" is used in
the context of the
present invention to refer to a compound that directs differentiation of PBMs
to macrophages of
the M1 type. The term "M2 inducer" is used in the context of the present
invention to refer to a
compound that directs differentiation of PBMs to macrophages of the M2 type.
The skilled person
is aware of a large number of ways to promote differentiation into either Ml
or M2 macrophages
The term "phagocytosis by macrophages" is the process by which a macrophage
engulfs a
solid particle to form an internal vesicle known as a phagosome.
Preferably the CD45- monocyte is not a dendritic cell, which differentiation
is controlled by
one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB,
PU/1, RBPJ,
IIRF4, and/or TCF4, and more preferably is not a dendritic cell.
In a preferred embodiment of the isolated targeted delivery system, the CD45+
leukocyte
cell is producible from a CD34+ hematopoietic precursor cell.
In a preferred embodiment of the of the isolated targeted delivery system
(i) the monocyte is a CD1 lb+ monocyte, preferably selected from the group
consisting of a
CD1 1b CD le monocyte, a CD1 1b CD16+ monocyte, a CD11b+ CD le CD16+ monocyte,
a CD1 1b CD14+ MHCII+ monocyte, a CD1113+ CD14+ CD1 15 monocyte, CD1113+
CD114+
monocyte, CD1 lb+ CD1 16 monocyte, CD1 1b CCR1+ monocyte, CD1 1b CCR2+
monocyte, CD1113+ CX3CR monocyte, CD1113+ CXR4+ monocyte, CD1113+ CXR6+
monocyte and a CD1 lb+ CD14+ CD33+ monocyte, preferably the monocyte is not a
dendritic
cell, which differentiation is controlled by one or more of the following
transcription factors:
IRF8, NFIL3, BATF3,RELB, PU/1, RBPJ, IMF4, and/or TCF4, and more preferably is
not
a dendritic cell.;
(ii) the differentiated monocyte or monocyte-macrophage is differentiated by M-
CSF and
selected from the group consisting of a macrophage, an activated macrophage,
preferably a
CD1 1b macrophage, more preferably a CD1113+ CD16+ macrophage, CD1 lb+ CD32+
macrophage, CD1 1b CD64+ macrophage, CD1 1b CD68" macrophage, preferably a
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37
CD1113+ CD86+ M1 macrophage, preferably producing inducible nitric oxide
synthetase
(iNOS) and/or secreting interleukin 12 (IL-12) or preferably CD1 1b CCR2" M2
macrophage, CD1 ib CD204 M2 macrophage, CD1 lb CD206" M2 macrophage, CD1 lb"
CD204" CD206+ M2 macrophage, CD1 1b Major Histocompatibility Complex II
(MLICIr) (low or hi expression) M2 macrophage, CD1 lb+ CD200R+ M2 macrophage,
CD1 1b+ CD163+ M2 macrophage or activated macrophage producing and/or
secreting
Arginase-1 and/or interleukin 10 (1L-10); preferably the differentiated
monocyte is not a
foam cell expressing Lectin-like oxidized low-density lipoprotein receptor-1
(Loxl+), C-X-
C chemokine receptor type 7 (CXCR7") and Nuclear factor (erythroid-derived 2)-
like 2
(NRF2+). A foam cell is a type of macrophage that localize to fatty deposits
on blood vessel
walls, where they ingest low-density lipoproteins and become loaded with
lipids giving them
a foamy appearance. These cells secrete various substances involved in plaque
growth and
their death promotes inflammation, thereby contributing to cardiovascular
disease;
(iii) monocyte-macrophage or activated monocyte-macrophage is differentiated
by M-CSF and
is preferably expressing at least one chemokine receptor, preferably selected
from the group
consisting of CCR1, CCR2, CXCR4, and CXCR6, or at least one growth factor
receptor,
preferably selected from the group consisting of macrophage colony stimulating
factor
Receptor (CD1 15), granulocyte colony stimulating factor Receptor (CD1 14),
and
granulocyte-macrophage colony stimulating factor Receptor (consisting of CD1
16 and
CD131); monocytes of these characteristics are particular suitable to treat
inflammatory
conditions and cancer;
(iv) the lymphocyte is selected from the group consisting of a CD3+ and CD4"
or CD8" T
lymphocyte, or a CD19", CD20", CD21", CD19+ CD20", CD19+ CD21", CD20" CD21",
or
CD19" CD20+ CD21+ B lymphocyte; or a natural killer (NK) cell, preferably the
INK cell is
selected from the group consisting of CD56" and without CD3 expression, or
CD16+CD56+,
CD56+CD94', CD56+CD158a", CD56'CD158r-, CD56+CD314k, CD56+CD335' cell; or
(v) the granulocyte is selected from the group consisting of a neutrophil,
preferably a CD66b
neutrophil, an eosinophil and a basophil, preferably a CD193+ eosinophil.
In a preferred embodiment of the isolated targeted delivery system the
activated
macrophage:
(i) is producible by in vitro incubation of a monocyte or macrophage
or their precursors with a
factor capable of altering expression markers on macrophages, preferably
(a) with at least one M1 inducer,
(b) with at least one M2 inducer,
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38
(c) or with a factor capable of altering the macrophages ability to secrete
cytokines,
preferably IL-10 and IL-12, chemokines and/or to produce iNOS, arginase or
other
immunomodulating enzymes; examples of such factors are: activated platelets,
IL-4, IL-
10, IL-13, immune complex of an antigen and antibody, IgG, heat activated
gamma-
globulin, glucocorticosteroid, tumour growth factor-I3 (TGF-f3), IL-1R, CC-
chemokine
ligand 2 (CCL-2), IL-6, Macrophage colony-stimulating factor (M-CSF),
peroxisome
proliferator-activated receptor y (PPARy) agonist, leukocyte inhibitory factor
(LIE),
adenosine, helminth and fungal infection, lipopolysaccharide (LPS), interferon
7 (INF-
7), viral and bacterial infection; in this respect it was observed that
activation of a
monocyte with a M1 inducer, particularly LPS will cause cell to express iNOS,
that
activation of a monocyte with a M1 inducer, particularly LPS will cause cell
not to
express Arginase-1, that activation of a monocyte with a M2 inducer,
particularly IL-4
will cause cell to express Arginase-1, and that activation of a monocyte with
a M2
inducer, particularly IL-4 will cause cell not to express iNOS,
(ii) is characterized by expression of at least one of following antigens:
CD64, CD86, CD16,
CD32, high expression of WWII, and/or production of iNOS and/or IL-12;
(iii) is producible by in vitro incubation of a monocyte or macrophage with a
factor capable of
inducing the ability of the macrophage to phagocytose, e.g. IL-18, opsonins
(for example
complement-derived proteins such as iC3b, immunoglobulin G), calcitonin gene-
related
peptide (CGRP), lipopolysaccharide (LPS), interferon y (INF-7)õ viral
infection and/or
bacterial infection;
(iv) is characterized by expression of at least one of following antigens:
CD204, CD206,
CD200R; CCR2, transferrin receptor (TfR), CXC-motive chemokine receptor 4
(CXCR4),
CD163, and/or T cell immunoglobulin-domain and mucin-domain 2 (TIM-2), and/or
show
low expression of WWII; activated macrophages having these properties are
particularly
suitable for complexes comprising ferritin as the iron binding protein;
(v) has the ability to phagocytose; and/or
(vi) is capable of cytokine secretion, preferably of IL-12, or IL-10, or
production of inducible
nitric oxide synthetase (iNOS) (or other pro-inflammatory compounds), arginase
or other
immunosuppressive/anti-inflammatory compounds.
In a preferred embodiment of the isolated targeted delivery system the M1
inducer for
differentiating macrophages into M1 macrophages is selected from the group
consisting of
lipopolysaccharide (LPS), interferon y (INF-7), and viral and bacterial
infection and the M2
inducer for differentiating macrophages into M2 macrophages is selected from
the group
consisting of IL-4, IL-10, IL-13, immune complex of an antigen and antibody,
IgG, heat activated
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39
gamma-globulin, glucocorticosteroid, tumour growth factor-13 (TGF-13), IL-1R,
CC-chemokine
ligand 2 (CCL-2), IL-6, Macrophage colony-stimulating factor (M-CSF),
peroxisome proliferator-
activated receptor y (PPARy) agonist, leukocyte inhibitory factor (LW),
adenosine, helminth and
fungal infection.
In a preferred embodiment of the targeted delivery system of the present
invention the
monocyte-macrophage:
(i) is producible from a CD34+ hematopoietic precursor cell;
(ii) is producible by in vitro incubation of monocytes with at least one
inducer, preferably M1
or M2 inducer, more preferably at least one M2 inducer;
(iii) is characterized by expression of at least one of the following
antigens: Tflt, CD163, TIM-
2, CD14, CD16, CD33, and/or CD115;
(iv) is characterized by expression of at least one of the following antigens:
TfR, CD163, TIM-
2, CXCR4, CD14, and/or CD16; and/or
(v) has the ability to phagocytose; and/or
(vi) is not a dendritic cell which differentiation is controlled by one or
more of the following
transcription factors: IRF8, NFIL3, BATF3 or RELB, PU/1, RBPJ, IRF4 or TCF4.
In this embodiment of the targeted delivery system of the present invention
the M1 inducer for
differentiating monocyte-macrophage cells is selected from the group
consisting of LPS,
INF-y or viral or bacterial infection or the M2 inducer for differentiating
monocytes is
selected from the group consisting of IL-4, IL-10, IL-13, immune complex of an
antigen and
antibody, IgG, heat activated gamma-globulins, Glueocorticostcroids, TGF-13,
IL-1R, CCL-
2, IL-6, M-CSF, PPARy agonist, Leukocyte inhibitory factor (LIF), cancer-
conditioned
medium, cancer cells, adenosine and helminth or fungal infection.
In a preferred embodiment of the targeted delivery system of the present
invention the
lymphocyte:
(i) is obtainable from blood, spleen, or bone marrow or is producible
from a CD34+ precursor
cell as known to the skilled person and also described in the, e.g. Lefort and
Kim, 2010, J
Vis Exp 40: 2017; Tassone and Fidler, 2012, Methods in Molecular Biology 882:
351-357;
Kouro et al. 2005, Current Protocols in Immunology, 66:F22F.1:22F.1.1-22F.1.9
;
(ii) is an immunologically competent lymphocyte;
(iii) expresses antigen specific T cell receptors; and/or
(iv) is characterized by expression of at least one of the following antigens:
(a) CD3 and CD4 or
CD8 or (b): CD19, CD20, CD21, CD19 CD20, CD19 CD21, CD20 CD21, or CD19 CD20
CD21 antigen, and is preferably capable of producing immunoglobulins
In a particularly preferred embodiment the CD45+ lymphocytes is a NK cell,
which
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(i) is obtainable from blood, spleen or bone marrow or producible from a
CD34r precursor cell;
and/or
(ii) is characterized by the lack of CD3 expression and expression of at least
one of the following
CD56+ and/or CD94-, CD158a CD158r CD314+ CD335 .
5 In a preferred embodiment of the targeted delivery system of the present
invention the granulocyte:
(i) is obtainable from blood, spleen or bone marrow or producible from a
CD34+ precursor cell
as described, e.g. in Kuhs etal. 2015, CurrProtocImmunol 111:7.23-1-7.23.16;
Coquery et
al. 2012, Cytometry A 81(9): 806-814; Swemydas and Lionakis 2013, J Vis Exp
77: 50586.;
(ii) is characterized by expression of at least one of the following CD66b
and/or CD193;
10 (iii) is a polymorphonuclear leukocyte characterized by the presence of
granules in its cytoplasm;
and/or
(iv) is characterized by expression of at least one of the following: TfR,
CD163, TIM-2, and/or
CXCR4.
The cell comprised in the isolated targeted delivery system may also be a
mesenchymal stem
15 cell. The term "mesenchymal stem cell" or "MSC" is used in the context
of the present invention
to refer to adult stem cells which are non-haematopoietic, multipotent stem
cells with the capacity
to differentiate into mesodermal lineage such as osteocytes, adipocytes and
chondrocytes as well
ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express
cell surface
markers like cluster of differentiation (CD)73, CD90, and CD105 and lack the
expression of CD45,
20 CD34, CD14/CD11b, CD19/CD20/CD79ct, and HLA (human leucocyte antigen)-DR.
Human
MSCs for the first time were reported in the bone marrow and till now they
have been isolated
from various tissues, including adipose tissue, placenta, amniotic fluid,
endometrium, dental
tissues, umbilical cord blood and umbilical cord tissue (Wharton's jelly).
They also have been
derived (i.e. differentiated) from Induced Pluripotent Stem Cells (iPSCs).
Thus, in a preferred
25 embodiment of the invention, the MSC is selected from the group
consisting of an umbilical cord
MSC, a bone marrow MSC, an adipose MSC, a placenta MSC, a dental MSC, an
amniotic fluid
MSC, an endometrium MSC, and an iPSC-derived MSC. Preferably, it is an
umbilical cord blood
MSC or an umbilical cord tissue (or Wharton's jelly) MSC. Furthermore, it is
preferred that the
MSC is a human MSC. The present inventors have observed that such MSCs stem
cells can be
30 loaded with the complex according to the invention and deliver it into
cancer cells.
In a seventh aspect the present invention relates to pharmaceutical or
diagnostic composition
comprising the polypeptide of the first aspect, the conjugate of the second
aspect, the complex of
the third aspect or the isolated targeted delivery system of the fourth aspect
and a pharmaceutically
acceptable carrier and/or suitable excipient(s).
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In instances where the pharmaceutical or diagnostic composition comprises
living cells, it
is preferred that carriers and excipients are chosen such as to keep the cells
alive.
"Pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a
state government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia
for use in animals, and more particularly in humans.
The term "carrier", as used herein, refers to a pharmacologically inactive
substance such as
but not limited to a diluent, excipient, surfactants, stabilizers,
physiological buffer solutions or
vehicles with which the pharmaceutically active substance is administered.
Such pharmaceutical
carriers can be liquid or solid. Liquid carrier include but are not limited to
sterile liquids, such as
saline solutions in water and oils, including but not limited to those of
petroleum, animal, vegetable
or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid carriers,
particularly for injectable solutions. A saline solution is a preferred
carrier when the
pharmaceutical composition is administered intravenously. Examples of suitable
pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by E. W.
Martin.
Suitable pharmaceutical "excipients" include starch, glucose, lactose,
sucrose, gelatine, malt,
rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried
skim milk, glycerol, propylene, glycol, water, ethanol and the like.
"Surfactants" include anionic, cationic, and non-ionic surfactants such as but
not limited to
sodium deoxycholate, sodium dodecylsulfate, Triton X-100, and polysorbates
such as polysorbate
20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.
"Stabilizers" include but are not limited to mannitol, sucrose, trehalose,
albumin, as well as
protease and/or nuclease antagonists.
"Physiological buffer solution" include but are not limited to sodium chloride
solution,
demineralized water, as well as suitable organic or inorganic buffer solutions
such as but not
limited to phosphate buffer, citrate buffer, tris buffer
(tris(hydroxymethyl)aminomethane), EIEPES
buffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3
morpholino-1
propanesulphonic acid). The choice of the respective buffer in general depends
on the desired
buffer molarity. Phosphate buffer are suitable, for example, for injection and
infusion solutions.
The term "adjuvant" refers to agents that augment, stimulate, activate,
potentiate, or
modulate the immune response to the pharmaceutically active substance
comprised in the
composition at either the cellular or humoral level, e.g. immunologic
adjuvants stimulate the
response of the immune system to the actual antigen, but have no immunological
effect
themselves. Examples of such adjuvants include but are not limited to
inorganic adjuvants (e.g.
inorganic metal salts such as aluminium phosphate or aluminium hydroxide),
organic adjuvants
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(e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's complete
adjuvant and Freund's
incomplete adjuvant), cytokines (e.g. IL-113, IL-2, IL-7, IL-12, IL-18, GM-
CFS, and INF-y)
particulate adjuvants (e.g. immuno-stimulatory complexes (ISCOMS), liposomes,
or
biodegradable microspheres), virosomes, bacterial adjuvants (e.g.
monophosphoryl lipid A, or
muramyl peptides), synthetic adjuvants (e.g. non-ionic block copolymers,
muramyl peptide
analogues, or synthetic lipid A), or synthetic polynucleotides adjuvants (e.g.
polyarginine or
polylysine).
As indicated above, the term "CD45+ leukocyte cell" is used throughout this
specification to
refer to a CD45+ monocyte, CD45+ monocyte-macrophage, CD45+ lymphocyte and/or
CD45 .
Preferably, the monocyte is not a dendritic cell, which differentiation is
controlled by one or more
of the following transcription factors: IRF8, NFIL3, 13ATF3, RELB, PU/1, RBPJ,
IIRF4, and/or
TCF4, and more preferably is not a dendritic cell Preferred subpopulations in
these general
categories of leukocytes are defined in the following by structural
parameters, e.g. presence or
absence of a given protein, functional properties and/or method of their
production/differentiation
As outlined above, the targeted delivery system of the present invention still
provides the
advantages outlined above, if in a population of cells not every cell has a
particular property in as
long as the majority of cells within that population has that property. Thus,
in the following the
property of one preferred cell of the targeted delivery system of the present
invention is described.
It is appreciated by the skilled person that a pharmaceutical composition of
the present invention
will comprises millions of cells and that not every cell within the population
will have the
functional and/or structural properties outlined herein but that the
pharmaceutical composition can
nevertheless be used to treat a disease, if the majority of cells share the
respective functional and/or
structural properties.
The cells comprised in the targeted delivery system, in particular CD45+
leukocyte cells or
MSCs, originate from the patient to be treated. In such case the cell loaded
with the complex would
be autologous to the patient. It is also envisioned that patients are MHC
typed prior to treatment
with the targeted delivery system of the present invention and that the cell
type used for a given
patient is MEW matched to the patient. In these two preferred embodiments the
cell is a primary
cell or derived by a low number of differentiation steps from a primary cell.
Alternatively, the cell
may be from an immortalized but preferably non-transformed cell line.
The blood used for isolation of CD45+ leukocyte cells, i.e. CD45+monocyte,
CD45+
monocyte-macrophage, CD45+ granulocyte, or CD45 1ymphocyte, in particular
CD45+ NK cell, is
preferably obtained from the patient to be treated or from a healthy donor.
Alternatively the blood
can be obtained from the blood bank. Use of umbilical cord blood is also
considered herein.
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In an eight aspect the present invention relates to the polypeptide of the
first aspect, the
conjugate of the second aspect, the complex of the third aspect or the
isolated targeted delivery
system of the fifth aspect for use in medicine.
In a ninth aspect the present invention relates to the polypeptide of the
first aspect, the
conjugate of the second aspect, the complex of the third aspect or the
isolated targeted delivery
system of the fifth aspect for use in treating, preventing and diagnosing a
tumour, preferably a
solid tumour and/or its metastases, preferably breast cancer, pancreatic
cancer, bladder cancer,
lung cancer, colon cancer, or a tumour having hypoxic areas; an inflammatory
disease or ischemic
areas, in particular in skin wounds or after organ infarctus (heart) or
ischemic retina; or for
prophylactic or therapeutic vaccination, in particular to prevent or treat an
infectious disease or
cancer. This aspect also includes targeted delivery of antigens to
physiological or non-
physiological lymph nodes in order to vaccinate an individual or to induce
immune memory.
In a tenth aspect the present invention relates to method of treating,
preventing or diagnosing
a tumour, preferably a solid tumour and/or its metastases, preferably breast
cancer, pancreatic
cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, liver
cancer,
glioma/glioblastoma or a tumour having hypoxic areas; an inflammatory disease
or ischemic areas,
in particular in skin wounds or after organ infarctus (heart) or ischemic
retina; or a method of
prophylactic or therapeutic vaccination, in particular to prevent or treat an
infectious disease or
cancer by administering an effective amount of the polypeptide of the first
aspect, the conjugate
of the second aspect, the complex of the third aspect or the isolated targeted
delivery system of the
fifth aspect to a subject in need thereof. This aspect also includes targeted
delivery of antigens to
physiological or non-physiological lymph nodes in order to vaccinate an
individual or to induce
immune memory.
The term "treatment" as used herein includes all types of preventive and/or
therapeutic
interventions medically allowed for the purpose of cure, temporary remission,
prevention, etc. for
different purposes including delaying or stopping the progress of a disease,
making a lesion regress
or disappear, preventing onset, or inhibiting recurrence.
Brief Description of Drawings
Fig. 1: In silico analysis of mutants: Hot spot prediction results by using
PredHS2for the human
H-Ferritin-TfR1 complex and FoldX for the human H-Ferritin-DNA virtual
complex. True
positives, common for both DNA binding and TfR1 binding are represented as
CPK.
Fig. 2: Sensorgrams corresponding to the interaction between the immobilized
his-tagged TFRC
receptor and human ferritin. Panel A: mutant Q1 1E, panel B: wild type. X-
axis: time (s).
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The same amount of receptor was trapped onto the chip surface (see methods
section for
details). Five different analyte concentrations (0.0625, 0.125, 0.250, 0.5 and
1mg/m1) were
used. For all analyte concentrations measured, the amount of ferritin bound is
higher for
the Q11E mutant than for wild-type ferritin. The global fit using a simple 1:1
binding mode
indicates a higher affinity and correspondingly a lower KD value.
Fig. 3: Native gel analysis of wild type ferritin and mutants showing that
wild type ferritin mainly
forms of double glued frames, aggregates of cages and larger forms of
aggregates,
however, mutations decrease the ability of ferritin to form aggregates,
therefore the mutant
ferritin variants are present as 24-mers (homogeneous cages).
Lane 1: Ferritin wild type-dox concentrated on 10 kDa Amicon.
Lane 2: Ferritin wild type-dox concentrated on 100 kDa Amicon
Lane 3: Q11E mutant-dox concentrated on 10 kDa Amicon.
Lane 4: QUE mutant-dox concentrated on 100 kna Amicon.
Lane 5: Q1 lE - Q15E mutant-dox concentrated on 10 kDa Amicon.
Lane 6: Q1 lE - Q15E mutant-dox concentrated on 100 kDa Amicon.
Fig. 4: Native gel analysis of wild type ferritin and mutants showing that
storage conditions did
not adversely affect the stability of the cages in the mutants, and the mutant
ferritin variants
are still present as 24-mers (homogeneous cages) after storage.
Lane 1: Q1 1E mutant-dox concentrated on 10 kDa Amicon.
Lane 2: Q11E mutant-dox concentrated on 100 kDaAmicon.
Lane 3: Q11E - Q15E mutant-dox concentrated on 10 kDa Amicon.
Lane 4: Q1 lE - Q15E mutant-dox concentrated on 100 kDa Amicon.
Lane 5: Ferritin wild type-dox concentrated on 10 kDa Amicon.
Lane 6: Ferritin wild type-dox concentrated on 100 kDa Amicon.
Fig. 5: Graphical representation of the calculation of doxorubicin loading
efficiency for Ft wild
type and Ft mutants. The average particle numbers per cage along with the
median are
marked on the graph.
Fig. 6: UV-Vis spectrum of Ft wild type and Q11E mutant after doxorubicin
encapsulation. The
initial concentration for both proteins was the same and equal 28,5 mg/ml. The
final
concentrations for Ft wild type and Q1 lE mutant were 10,2 mg/ml and 28,5
mg/ml. Each
concentration and the recorded spectra are for a volume of 1 ml protein
solution. The
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extinction coefficient for Ft wild type and Q1 1E mutant are the same and
equal 18600 M-
lcm-1 = 278 nm).
Fig. 7: Graphs of tumour cell viability after 72 h co-culture with
macrophages. The concentration
5
of ferritin cages filled with doxorubicin was the same for each variant of
ferritin and was
equal to 1 mg/ml.
Fig. 8: Picture of gel showing RNA association with Q1 lE ferritin mutant that
is not observed in
case of wild-type protein (wt).
Fig. 9. The LC-MS spectrum of ferritin after conjugation with vcMMAE The
spectrum shows
that it is mostly a ferritin fraction with two drug molecules attached. The
molar drug to
protein ratio of the conjugate is equal to 1.95
Example Section
Example 1¨ In silico analysis of mutants
Identification of small portions of a protein-protein interface that
contribute to the majority
of the binding free energy can provide crucial information for understanding
the nature of the
interaction and recognition properties. These portions are referred to as "hot
spots" in recent
computational chemistry approaches (Hao Wang, et al., Sci. Rep. 8, 14285
2018). Here, the
inventors describe the application of the PredHS2 software
(http://predhs2.denglab.org) coupled
to MD minimization in order to predict hot spots from the complex of human H
chain ferritin and
Transferrin receptor (PDB ID:6H51). Based on PredHSmethod (Wei, L, et Al.
Comb.chemistry &
high throughput screening 19, 144-152 2016), the inventors built a dataset of
14 interface residues
on H Ferritin interface that corresponds to the contacts obtained from the
CD71/H-ferritin complex
recently identified by Montemiglio et al., (Montemiglio et al., 2019 Nat Comm
10 1121-1121).
Then the inventors generated a set of 476 sequences (single mutants of the 14
positions) obtained
after removal of redundant and irrelevant sequences utilizing a two-step
feature selection method,
which consists of a minimum Redundancy Maximum Relevance (mRIVIR) procedure
and a
sequential forward selection process that eliminates all mutations that are
considered not
compatible with folding properties (e.g. Gly or Pro within alpha helices
regions). Thereafter,
energy minimization of the relevant structure, exposure to solvent and energy
features, together
with Euclidean and Voronoi neighbourhood properties was carried out. In order
to assess the
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performance of the prediction model, the inventors adopted 10-fold cross-
validation together with
commonly used measures, such as specificity (SPE), precision (PRE),
sensitivity (SEN/Recall),
accuracy (ACC), Fl-score (F1) and Matthews correlation coefficient (MCC). Data
relative to the
best energy matches (ZAPP) and measures are listed in Table 1. As apparent,
the isosteric
glutamine substitutions with a glutamate residue predicted a higher binding
free energy
contribution whereas non-isosteric mutations (even when bearing the same
charge) invariably led
to a decrease in binding free energy contribution (RM SE). Apparently, Voronoi
contribution
(polynomial squared distances minimization), played a key role in contributing
to the binding free
energy as it confers energy penalties to the voids generated by missing atoms
in non-isosteric
mutants, or even higher gaps in the case of bulkier residues. As shown in Fig.
1, four hot spots (8,
11, 12 andl 5) have been experimentally determined at the binding interface.
These residues were
individually taken into account. Multiple mutants have not been considered in
these calculations
as the resulting binding free energies appeared to be unrealistically high.
TABLE I - Prediction of "hot spots" for the complex of human H chain ferritin
and
Transferrin receptor.
The four residues in positions 8, 11, 12 and 15 (PDB ID:6H5I) were found to be
the most
important contributors to the H-ferritin / CD71 receptor complex formation.
Here the inventors
show that individual, isosteric mutations of these "hot spots" provide a
further binding free energy
gain to the complex.
Mutant Composition of ZAPP and Performance of Energy Functions with
Terms
RMSE
(Kcal mol- ACC SPE PRE SEN Fl
MCC
Q8E 7.2 0.89 0.91 0.85 0.78 0.79
0.70
Q1 1E 8.1 0.87 0.95 0.84 0.83 0. 80
0.70
N12D 4.9 0.91 0.93 0.85 0.81 0.79
0.70
Q15E 5.3 0.94 0.94 0.86 0.87 0.82
0.70
WT 4.3 0.88 0.94 0.86 0.79 0.80
0.70
A second algorithm has been applied based on Protein-assisted DNA assembly
(PADA1)
algorithm to predict and model the binding of double-stranded DNA (dsDNA) to
proteins. PADA1
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includes an empirical interaction model generator in combination with an ultra-
fast statistical
knowledge-based force field, which act in synergy in order to perform dsDP
docking (Blanco JD,
et al., Nucleic Acids Res. 2018 May 4;46(8):3852-3863).This algorithm uses
fragment pairs
(peptide paired to short dsDNA) that represent empirical, compatible backbone
conformations
found in nature. DNA¨protein structures modeled by PADAI have been used in
combination with
FoldX (protein design software) to predict DNA recognition sequences. The
cooperative action
between PADAland FoldX, for side chain refinement and interface optimization,
turns ModelX
in a powerful modelling tool for predicting key residues at the core of
ferritin-DNA interaction. In
the case of Human H ferritin, we measured the atomic "all-to-all" distances
between the protein
fragment and the corresponding dnaX fragment. Then, using the atomic distance
distributions, we
obtained the statistical parameters (mean and standard deviation of the
distances) for all possible
contacts between the protein and dsDNA fragments included in the interaction
database. All-to-
all distances between contacting nucleotide-amino acid pairs were measured
according to the
limits suggested by Blanco JD et al , (a contact is considered when at least
one atom of the amino
acid, including side chains, is less than 4 A from any atom in the
nucleotide). Most interestingly,
7 residues were found to be responsible for nucleic acid binding properties,
which comprise the 4
glutamines already demonstrated to contribute to the receptor binding
interface plus glutamine 83
together with lysines 86 and 87 (see Fig. 1).
Example 2¨ In vitro binding to ferritin receptor
Binding of wild type and QI IE mutant ferritin to TfRI was analysed by surface
plasmon
resonance.
Examples 3-8
The inventors generated further ferritin variants based on the Q1 1E and Q11E-
Q15E mutants by
adding the mutations K54E, K72E, K87Q, K144E, C91S and C103S. Mutation C131S
was further
added to mutant Q1 1E. The properties of these ferritin variants were further
analysed in examples
3-8.
Example 3
Native PAGE gel has been performed in order to check the size of the protein
after encapsulation
and purification. It clearly shows that wild type protein shows a
heterogeneity of forms, in addition
to cages it contains aggregates of cages and larger forms of aggregates, in
contrary to mutated
protein, which is are present as 24-mers (homogeneous cages). A greater degree
of aggregation
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48
adversely affects the loading efficiency and protein recovery after loading
with doxorubicin (Fig.
3).
Example 4
Storage conditions for wild type ferritin are limited. Storage in a freezer (-
80 degrees Celsius) and
thawing causes an increase of cages aggregation, which prevents their
separation on native
electrophoresis. In contrast, the Q11E mutant reduces the presence of
aggregates and allows the
storage of ferritin cages with doxorubicin in the freezer (Fig.4).
Example 5
The calculation of the encapsulation efficiency indicates that Ft mutants Q11E
and Q11E - Q15E
are able to load on average more doxorubicin molecules into their cages
compared to Ft wild type,
55, 63, 23 molecules, respectively (Fig. 5).
Example 6
UV-Vis spectrum analysis has shown that protein recovery after loading was
100% for mutant
Q11E and only 38% for wild ferritin (Fig.6).
Example 7
In vitro experiments have shown that ferritin mutants reveal better
cytotoxicity against tumor cells.
The doxorubicin packed proteins were inserted into macrophages and then co-
cultured with breast
and ovarian cancer cell lines: AIDA-MB 231, Skov3 and 4T1. The number of
viable cells after co-
culture was the lowest for the Q11E-Q15E mutant (Fig. 7).
Example 8
1 ml reaction containing 2mg Ft and 4ug siRNA in DPBS, pH lowered to values as
indicated (2.5,
3.2, 4.4, 5.6 and 68, respectively). Samples were incubated 15 min in RT, then
Na0II was added
to adjust pH to neutral (pH 7). Non-associated free siRNA was removed by 4
centrifugation steps
on Amicons with 100 kDa cut-off. As shown in Fig. g, Ferritin Q11E mutant
associated with
siRNA when incubated in pH range 4.4 to 6.8 (arrow), but not in 2.4 and 3.2,
conditions in which
Ferritin cage is likely to be disrupted. This observation suggests that Ft
nanocage integrity might
be crucial for observed association. In contrast, no association with siRNA
was observed for wild
type Ferritin variant, independently from pH conditions during Ft incubation
with siRNA.
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Example 9
Human heavy chain ferritin can be covalently linked to host hydrophobic drug
molecules within
the cysteine residues. Maleimide functionalized drugs, such as a tubulin
inhibitor Monomethyl
Auristatin (MMAE) is one of the most notable examples of potent cytotoxic that
can be readily
and specifically attached. The inventors have conjugated this drug to ferritin
according to the
following procedure: The auristatin E analogue, maleimidocaproyl-valine-
citrulline-p-
aminobenzoyloxycarbonyl-monomethyl auristatin E (voMMAE) was obtained from
MedChem
Express (Princeton, NJ). The ferritin vcM1VIAE adduct was prepared as follows:
Human heavy
chain ferritin according to SEQ ID NO: 77 was used. Ferritin solution was
adjusted to a
concentration of 125 [tM with reaction buffer (20 mM HEPES ((4-(2-
hydroxyethyl)-1-
piperazineethanesulfonic acid)), 0.04 % Polysorbate 80, pH 7.0) and conjugated
with 5-fold molar
excess of voMMAE at room temperature at 4 C for 4 hours. Maleimide groups
react efficiently
and specifically with free (reduced) sulfhydryls at pH 6.5-7.5 to form stable
thioether bonds. The
final conjugate was dialyzed in washing buffer (20 mM HEPES ((4-(2-
hydroxyethyl)-1-
piperazineethanesulfonic acid)), 002 % Polysorbate 80, 2 % Glycerin, pH 7.0),
to remove
unbound vcMMAE, and concentrated at Amicon centrifugal filter device. The
molar drug to
protein ratio of the obtained conjugate was determined by LC-MS analysis and
it was equal to 1.95
(see Fig. 9). The concentration of Ft-voMMAE conjugate was determined by BCA
colorimetric
assay based on the absorbance at 562 nm.
Items
1. A ferritin variant polypeptide, wherein one or more cysteine residues,
in particular cysteine
residues at position 9 I , 103 and/or 1 3 I indicated with respect to SEQ ID
NO. I, are deleted
or substituted, preferably substituted with serine residues.
2. The ferritin variant polypeptide of item 1, wherein the ferritin variant
polypeptide has a
sequence according to SEQ ID NO. 82, SEQ ID NO. 1 or SEQ ID NO. 2, wherein at
least
one, preferably all, cysteine residues at position 91, 103 and/or 131 are
deleted or
substituted, preferably substituted with serine residues, and wherein the
sequences
according to SEQ ID NO. 82, SEQ ID NO. 1 and SEQ ID NO. 2 may further comprise
1-
5, 1-10, 1-15, 1-20 or 1-25 amino acid mutations outside position 91, 103
and/or 131.
3. The ferritin variant polypeptide of item 1 or 2, wherein one, two, three
or four, preferably
four, lysine residues, preferably lysine residues at position 54, 72, 87
and/or 144 indicated
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with respect to SEQ ID NO. 1 (human wild-type heavy chain ferritin), are
deleted or
substituted with a non-basic amino acid, preferably E or Q, most preferably
wherein K54
is substituted with E, K72 is substituted with E, K87 is substituted with Q
and K144 is
substituted with E.
5
4. The ferritin variant polypeptide of any one of items 1 to 3, wherein the
ferritin variant
polypeptide has a sequence according to SEQ ID NO. 75 or SEQ 11) NO. 76 or a
sequence
according to SEQ ID NO. 75 or SEQ ID NO. 76 comprising 1-5, e.g. 1, 2, 3, 4 or
5, or 1-
10, e.g. I, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations outside position
91, 103 and/or
10 131.
5. The ferritin variant polypeptide of any one of items 1 to 4, further
comprising a transferrin
receptor binding domain (TRBD) of a ferritin variant wherein the TRBD in
comparison to
the wild-type ferritin on which it is based comprises one or more glutamine
residues
15 mutated into glutamic acid residues and/or one or more
asparagine residues mutated into
aspartic acid residues, wherein in particular at least one, preferably all
mutations are
comprised in the 20 N-terminal amino acids of the wild-type ferritin.
6. The ferritin variant polypeptide of item 5, wherein the TRBD comprises
at least the
20 following amino acid sequence:
MTTASX1SZVRZBYHZDX2EAA (SEQ ID NO. 3)
Xi = S or T, preferably T;
25 X2= S or A, preferably S;
Z = Q or E; and
B = N or D;
wherein at least one Z or B is E or D,
which may further comprise one, two or three amino acid substitutions outside
Z and/or B,
30 and wherein the M at position 1 may be present or absent.
7. The ferritin variant polypeptide according to item 5 or 6, wherein the
TRBD comprises at
least an amino acid sequence selected from the group comprising SEQ ID NO. 04
to SEQ
ID NO. 63, particularly from the group consisting of SEQ ID NO. 05, 11, 12,
15, 20, 26,
35 27, 30, 35, 41, 42, 45, 50, 56, 57 and 60, more particularly
from the group consisting of
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SEQ ID NO. 05, 12, 20, 27, 35, 42, 50 and 57, which may further comprise one,
two or
three amino acid substitutions outside amino acid positions 8, 11, 12 and/or
15, and
wherein the M at position 1 may be present or absent.
8. The ferritin variant polypeptide according to any of items 5 to 7,
wherein the affinity of the
TRBD to Tflt-1 is increased in comparison to the TRBD of the wild-type
ferritin at least
(>) 1.5x, > 2x, > 3x, > 4x,? 5x, > 10x, > 20x, > 30x, > 40x, > 50x, but less
than (<) 100x,
< 75x < 50x, < 40x, < 30x, < 20x, < 10x, or < 5x, in particular the affinity
of the TRBD to
TfR-1 is increased between 1.5x - 50x, 2x - 50x, 3x - 50x, 4x - 50x, 5x - 50x,
10x - 50x,
20x - 50x, 30x - 50x, 40x - 50x, 1.5x - 10x, 2x - 20x or 5x - 30x in
comparison to the
TRBD of the wild-type ferritin.
9. A nucleic acid encoding the polypeptide of any of items 1 to 8.
10. A vector comprising the nucleic acid of item 9.
11. A conjugate comprising the polypeptide of items 1 to 8 and at
least one label and/or at least
one drug.
12. A complex comprising at least one polypeptide of items 1 to 8 and/or at
least one conjugate
of item 16.
13. The complex of item 12 further comprising at least one label
and/or at least one drug.
14. The conjugate of item 11 or the complex of items 12 or 13, wherein the
label is selected
from the group consisting of
a. a fluorescent dye, in particular a fluorescent dye selected from the
group consisting of
the following classes of fluorescent dyes: Xanthens, Acridines, Oxazines,
Cynines,
Styryl dyes, Coumarines, Porphines, Metal-Ligand-Complexes, Fluorescent
proteins,
Nanocrystals, Perylenes and Phtalocyanines as well as conjugates and
combinations
of these classes of dyes;
b. a radioisotope/fluorescence emitting isotope, in particular a
radioisotope/fluorescence
emitting isotope selected from the group consisting of alpha radiation
emitting
isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes,
X-ray
emitting isotopes, fluorescent isotopes, such as 65Tb, fluorescence emitting
isotopes,
such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 140La, 175Yb, 153Sm,
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166Ho,88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru,
109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 1231, 1241, 1311, 197Hg, 211At, 169Eu,
203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag as well as conjugates
and combinations of above with proteins, peptides, small molecular inhibitors,
antibodies or other compounds;
c. a detectable polypeptide, in particular an autofluorescent protein,
preferably green
fluorescent protein or any structural variant thereof with an altered
adsorption and/or
emission spectrum or nucleic acid encoding a detectable polypeptide; and
d. a contrast agent, in particular a contrast agent comprising a paramagnetic
agent,
preferably selected from Gd, Eu, W and Mn, or ferrihydride.
15. The conjugate of item 11 or 14 or the complex of items 12 to 13,
wherein the drug is
selected from the group consisting of an anticancer drug, in particular a
cytostatic drug,
cytotoxic drug or prodrug thereof, an anti-arteriosclerotic drug, and an anti-
inflammatory
or immunomodulatory drug.
16. The conjugate of item 11, 14 or 15 comprising a drug, wherein the drug
is auristatin, in
particular monomethyl auristatin (MMAE), conjugated to the polypeptide via a
maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl linker.
17 An isolated targeted delivery system comprising a cell, wherein
the cell comprises the
polypeptide of items 1 to 8, the conjugate of item 11 or 14 to 16, or the
complex of item
12 to 16, wherein particularly the cell is a CD45+ leukocyte, more
particularly a CD45+
leukocyte selected from the group consisting of a monocyte, a differentiated
monocyte,
lymphocyte and a granulocyte.
18. A pharmaceutical or diagnostic composition comprising the polypeptide
of items 1 to 8,
the conjugate of item 11 or 14 to 16 or the complex of item 12 to 16 or the
isolated targeted
delivery system of item 17 and a pharmaceutically acceptable carrier and/or
suitable
excipient(s).
19. The polypeptide of items 1 to 8, the conjugate of item 11 or 14 to 16
or the complex of
item 12 to 16 or the isolated targeted delivery system of item 17 for use in
medicine.
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