U.S. patent application number 16/312726 was filed with the patent office on 2019-10-31 for cellular targeted pharmaceutically active substance or label delivery system.
The applicant listed for this patent is CELLIS AG. Invention is credited to Paola BAIOCCO, Irene BENN, Alberto BOFFI, Lukasz KIRAGA, Magdalena KROL, Zofia PLICH, Tomasz RYGIEL, Aleksandra SZULC, Bartlomiej TACIAK, Katarzyna TONECKA, Katarzyna ULEWICZ.
Application Number | 20190328911 16/312726 |
Document ID | / |
Family ID | 56235808 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190328911 |
Kind Code |
A1 |
KROL; Magdalena ; et
al. |
October 31, 2019 |
CELLULAR TARGETED PHARMACEUTICALLY ACTIVE SUBSTANCE OR LABEL
DELIVERY SYSTEM
Abstract
The present invention relates to an isolated cellular targeted
delivery system comprising a CD45+ leukocyte cell comprising within
said cell a complex of one or more iron binding proteins and an
active pharmaceutically active substance and/or label as well as
methods for producing such isolated cellular targeted delivery
system and uses of such system for prophylaxis, therapy, diagnosis
or theragnosis, in particular for prophylactic or therapeutic
vaccination, therapy of cancer, particularly metastatic cancer or
inflammatory diseases.
Inventors: |
KROL; Magdalena; (Warsaw,
PL) ; BENN; Irene; (Rome, IT) ; BAIOCCO;
Paola; (Rome, IT) ; RYGIEL; Tomasz; (Warsaw,
PL) ; BOFFI; Alberto; (Rome, IT) ; SZULC;
Aleksandra; (Zurich, PL) ; PLICH; Zofia;
(Zurich, PL) ; TONECKA; Katarzyna; (Zurich,
PL) ; ULEWICZ; Katarzyna; (Zurich, PL) ;
TACIAK; Bartlomiej; (Zurich, PL) ; KIRAGA;
Lukasz; (Zurich, PL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELLIS AG |
Zurich |
|
PL |
|
|
Family ID: |
56235808 |
Appl. No.: |
16/312726 |
Filed: |
December 21, 2016 |
PCT Filed: |
December 21, 2016 |
PCT NO: |
PCT/PL2016/050057 |
371 Date: |
December 21, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 13/10 20180101;
C12N 2501/2313 20130101; C12N 2501/90 20130101; C12N 2501/21
20130101; A61K 38/42 20130101; A61P 11/00 20180101; A61K 45/06
20130101; A61K 47/64 20170801; C12N 5/0646 20130101; A61K 38/40
20130101; C12N 5/0636 20130101; A61P 31/00 20180101; A61P 35/00
20180101; A61K 35/17 20130101; C12N 2501/2304 20130101; A61K
51/1203 20130101; A61P 35/04 20180101; A61K 47/6901 20170801; A61K
47/6445 20170801; A61K 47/6811 20170801; A61P 9/10 20180101; A61P
15/00 20180101; C12N 2501/231 20130101; A61K 49/0056 20130101; A61K
49/1896 20130101; A61P 1/18 20180101; C12N 5/0642 20130101; A61P
27/02 20180101; A61P 29/00 20180101; C12N 5/0645 20130101; A61P
1/04 20180101; C12N 2501/15 20130101; A61K 51/0497 20130101; A61P
43/00 20180101; C12N 2501/999 20130101; A61K 35/15 20130101; A61P
37/02 20180101; A61K 47/644 20170801; A61P 17/02 20180101; C12N
2501/25 20130101; C12N 2501/24 20130101 |
International
Class: |
A61K 51/12 20060101
A61K051/12; A61K 47/64 20060101 A61K047/64; A61K 35/15 20060101
A61K035/15; A61K 35/17 20060101 A61K035/17; A61K 51/04 20060101
A61K051/04; A61K 47/69 20060101 A61K047/69; A61K 45/06 20060101
A61K045/06; A61K 47/68 20060101 A61K047/68; A61K 49/18 20060101
A61K049/18; A61K 49/00 20060101 A61K049/00; C12N 5/0786 20060101
C12N005/0786; C12N 5/0783 20060101 C12N005/0783; C12N 5/0787
20060101 C12N005/0787 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2016 |
EP |
PCT/EP2016/064483 |
Jun 22, 2016 |
EP |
PCT/EP2016/064484 |
Claims
1. An isolated targeted delivery system comprising a CD45.sup.+
monocyte, CD45.sup.+ monocyte-macrophage, CD45.sup.+ lymphocyte
and/or CD45.sup.+ granulocyte (commonly referred to as "CD45.sup.+
leukocyte cell") comprising within said cell a complex of one or
more iron binding proteins and a pharmaceutically active substance,
label or pharmaceutically active substance and label.
2. An isolated targeted delivery system comprising a CD45.sup.+
leukocyte cell comprising one or more label.
3. The isolated targeted delivery system according to claim 1,
wherein the CD45.sup.+ leukocyte cell is producible from a
CD34.sup.+ hematopoietic precursor cell.
4. The isolated targeted delivery system according to claim 3,
wherein (i) the monocyte is a CD11b.sup.+ monocyte, but is not a
dendritic cell which differentiation is controlled by following
transcription factors: IFN-regulatory factor 8 (IRF8), nuclear
factor 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); (ii) the differentiated monocyte is selected from
the group consisting of a macrophage, an activated macrophage,
preferably a CD11b.sup.+ macrophage, more preferably a CD11b.sup.+
CD16.sup.+ macrophage, CD11b.sup.+ CD32.sup.+ macrophage,
CD11b.sup.+ CD64.sup.+ macrophage, CD11b.sup.+ CD68.sup.+
macrophage, preferably a CD11b.sup.+ CD86.sup.+ M1 macrophage,
preferably producing iNOS and/or secreting interleukin 12 (IL-12)
or preferably CD11b.sup.+ CCR2.sup.+ M2 macrophage, CD11b.sup.+
CD204.sup.+ M2 macrophage, CD11b.sup.+ CD206.sup.+ M2 macrophage,
CD11b.sup.+ CD204.sup.+ CD206.sup.+ M2 macrophage, CD11b.sup.+
Mayor Histocompatibility Complex II.sup.+ (MHCII.sup.+) (low or hi
expression) M2 macrophage, CD11b.sup.+ CD200R.sup.+ M2 macrophage,
CD11b.sup.+ CD163.sup.+ M2 macrophage or activated macrophage
producing arginase and/or secreting interleukin 10 (IL-10);
preferably the differentiated monocyte-macrophage is not a
Lox1.sup.+, CXCR7.sup.+ and NRF2.sup.+ foam cell; (iii)
monocyte-macrophage or activated monocyte-macrophage expressing of
at least one chemokine receptor, or at least one growth factor
receptor; (iv) the lymphocyte is selected from the group consisting
of a CD3.sup.+ and CD4.sup.+ or CD8.sup.+ T lymphocyte, or a
CD19.sup.+, CD20.sup.+, CD21.sup.+, CD19.sup.+ CD20.sup.+,
CD19.sup.+ CD21.sup.+, CD20.sup.+ CD21.sup.+, or CD19.sup.+
CD20.sup.+ CD21.sup.+ B lymphocyte, and a natural killer (NK) cell;
or (v) the granulocyte is selected from the group consisting of a
neutrophil, an eosinophil and a basophil.
5. The isolated targeted delivery system of claim 4, wherein the
activated macrophage: (i) is producible by in vitro incubation of a
monocyte or macrophage with a factor capable of altering expression
markers on macrophages; (ii) is characterized by expression of at
least one of following antigens: CD64, CD86, CD16, CD32, high
expression of WICK 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; (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 MHCII; (v) has the ability to
phagocytose; and/or (vi) is capable of cytokine secretion, or
production of inducible nitric oxide synthetase (iNOS) or other
pro-inflammatory compounds, arginase or other
immunosuppressive/anti-inflammatory compounds.
6. The targeted delivery system according to claim 5, wherein: (i)
the M1 inducer is selected from the group consisting of LPS,
INF-.gamma., and viral and bacterial infection; or (ii) the M2
inducer is selected from the group consisting of IL-4, IL-10,
IL-13, immune complex of an antigen and antibody, IgG, heat
activated gamma-globulin, glucocorticosteroid, TGF-.beta., IL-1R,
CCL-2, IL-6, M-CSF, PPAR.gamma. agonist, Leukocyte inhibitory
factor, adenosine, helminth and fungal infection.
7. The isolated targeted delivery system of claim 4, wherein the
monocyte-macrophage cell: (i) is producible from a CD34.sup.+
hematopoietic precursor cell; (ii) is producible by in vitro
incubation of monocytes/monocyte-macrophage with at least one
inducer; (iii) is characterized by expression of at least one of
the following antigens: TfR.sup.+, CD163.sup.+, TIM-2.sup.+,
CD14.sup.+, CD16.sup.+, CD33.sup.+, and/or CD115.sup.+; (iv) is
characterized by expression of at least one of the following
antigens: TfR.sup.+, CD163.sup.+, TIM-2.sup.+, CXCR4.sup.+,
CD14.sup.+, and/or CD16.sup.+; and/or (v) has the ability to
phagocytose.
8. The targeted delivery system according to claim 7, wherein: (i)
the M1 inducer is selected from the group consisting of LPS,
INF-.gamma., or viral or bacterial infection; (ii) the M2 inducer
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, Glucocorticosteroids, TGF-.beta., IL-1R, CCL-2,
IL-6, M-CSF, PPAR.gamma. agonist, Leukocyte inhibitory factor,
cancer-conditioned medium, cancer cells, adenosine and helminth or
fungal infection.
9. The isolated targeted delivery system of claim 4, wherein the
lymphocyte: (i) is obtainable from blood, spleen, or bone marrow or
is producible from a CD34.sup.+ precursor cell; (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.sup.+
and CD4.sup.+ or CD8.sup.+ or (b): CD19.sup.+, CD20.sup.+,
CD21.sup.+, CD19.sup.+ CD20.sup.+, CD19.sup.+ CD21.sup.+,
CD20.sup.+ CD21.sup.+, or CD19.sup.+ CD20.sup.+ CD21.sup.+
antigen.
10. The isolated targeted delivery system of claim 4, wherein the
granulocyte: (i) is obtainable from blood, spleen or bone marrow or
producible from a CD34.sup.+ precursor cell; (ii) is characterized
by expression of at least one of the following CD66b.sup.+ and/or
CD193.sup.+; (iii) is a polymorphonuclear leukocyte characterized
by the presence of granules in their cytoplasm; and/or (iv) is
characterized by expression of at least one of the following:
TfR.sup.+, CD163.sup.+, TIM-2.sup.+, and/or CXCR4.sup.+.
11. The isolated targeted delivery system of claim 4, wherein the
NK cell: (i) is obtainable from blood, spleen or bone marrow or
producible from a CD34.sup.+ precursor cell; and/or (ii) is
characterized by the lack of CD3 expression and expression of at
least one of the following CD56.sup.+ and/or CD94.sup.+,
CD158a.sup.+ CD158f.sup.+ CD314.sup.+ CD335.sup.+.
12. The isolated targeted delivery system of claim 1, wherein the
iron binding protein is selected from the group consisting of
ferritin, haemoglobin, haemoglobin-haptoglobin complex, hemopexin,
transferrin, and lactoferrin.
13. The isolated targeted delivery system according to claim 1,
wherein the pharmaceutically active substance is selected from the
group consisting of a protein, a nucleic acid, a non-protein
non-nucleic acid compound with a molecular weight of less than 1.5
kD, an anti-arteriosclerotic drug, an anti-inflammatory drug, a
photosensitizing compound, a virus, and a .alpha. or radiation
emitting radioisotope, which also emit a cell damaging amount of
.gamma. radiation or a cell damaging amount of .alpha. radiation,
or a complex of the compound or isotope linked to a
nanoparticle.
14. The isolated targeted delivery system according to claim 13,
wherein the pharmaceutically active substance is an anticancer
drug, wherein 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 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 methylhydrazin derivative, an ene-diyne
antibiotic, a maytansinoid 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.
15. The isolated targeted delivery system according to claim 13,
wherein the pharmaceutically active substance is an anticancer
drug, wherein the anticancer drug is selected from the group
consisting of a acediasulfone, aclarubicine, ambazone,
aminoglutethimide, L-asparaginase, azathioprine, banoxantrone,
bendamustine, bleomycin, busulfan, calcium folinate, carboplatin,
carpecitabine, carmustine, celecoxib, chlorambucil, cis-platin,
cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin
dapsone, daunorubicin, dibrompropamidine, diethylstilbestrole,
docetaxel, doxorubicin, enediynes, epirubicin, epothilone B,
epothilone D, estramucin phosphate, estrogen, ethinylestradiole,
etoposide, flavopiridol, floxuridine, fludarabine, fluorouracil,
fluoxymesterone, flutamide fosfestrol, furazolidone, gemcitabine,
gonadotropin releasing hormone analog, hexamethylmelamine,
hydroxycarbamide, hydroxymethylnitrofurantoin,
hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine,
ifosfamide, interferon .alpha., irinotecan, leuprolide, lomustine,
lurtotecan, mafenide sulfate olamide, 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, phthalyl
sulfathiazole, pipobroman, prednimustine, prednisone, preussin,
procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib,
rosiglitazone, salazosulfapyridine, scriflavinium chloride,
semustine streptozocine, sulfacarbamide, sulfacetamide,
sulfachlopyridazine, sulfadiazine, sulfadicramide,
sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine,
sulfaguanole, sulfamethizole, sulfamethoxazole, co-trimoxazole,
sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole,
sulfanilamide, sulfaperin, 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, dynemycin A, maytansine,
melphalan, mertansine, and neocazinostatin.
16. The isolated targeted delivery system according to claim 13,
wherein the immunomodulatory drugs activate or inhibit activity of
immune cells, preferably the immunomodulatory drugs are ligands or
antagonists of Pattern Recognition Receptors, particularly
Toll-like Receptors, NOD-like receptors (NLR), RIG-I-like receptors
(RLR).
17. The isolated targeted delivery system according to claim 13,
wherein the pharmaceutically active substance is an anticancer
drug, wherein the anticancer drug is a proliferation inhibiting
protein, or an antibody or antibody like binding protein that
specifically binds to a proliferation promoting protein or a
nucleic acid.
18. The isolated targeted delivery system according to claim 1
comprising a pharmaceutically active substance, wherein the
pharmaceutically active substance is a hypoxia-activated
prodrug.
19. The isolated targeted delivery system according to claim 1
comprising a pharmaceutically active substance, wherein the
pharmaceutically active substance is an antigen or a nucleic acid
encoding an antigen.
20. The isolated targeted delivery system of claim 1 comprising a
label, wherein the label is selected from the group consisting of a
fluorescent dye, a fluorescence emitting isotope, a radioisotope, a
detectable polypeptide or nucleic acid encoding a detectable
polypeptide and a contrast agent.
21. The isolated targeted delivery system of claim 1 comprising a
label, wherein the label comprises a chelating agent which forms a
complex with divalent or trivalent metal cations.
22. The isolated targeted delivery system of claim 21, wherein 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,
Diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA) and
6-Hydrazinopyridine-3-carboxylic acid (HYNIC).
23. The isolated targeted delivery system of claim 20, wherein the
label comprises a contrast agent, wherein the contrast agent
comprises a paramagnetic agent.
24. The isolated targeted delivery system of claim 20, wherein the
label comprises a radioisotope/fluorescence emitting isotope,
wherein 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,
fluorescence emitting isotopes, as well as conjugates and
combinations of the above with proteins, peptides, small molecular
inhibitors, antibodies or other compounds.
25. The isolated targeted delivery system of claim 20, wherein the
label comprises a fluorescent dye, wherein the fluorescence 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.
26. The isolated targeted delivery system according to claim 20,
wherein the label comprises a detectable polypeptide, wherein the
detectable polypeptide is an autofluorescent protein.
27. The isolated targeted delivery system according to claim 1,
wherein: (i) the bond(s) between the iron binding protein(s) and
the pharmaceutically active substance, label or pharmaceutically
active substance and label comprised in the complex are covalent
and/or non-covalent; and/or (ii) the pharmaceutically active
substance, label or pharmaceutically active substance and label
comprised in the complex is entrapped/encapsulated by the iron
binding protein or multimers thereof.
28. Method of preparation of the isolated targeted delivery system
of claim 1 comprising steps of a) providing purified iron binding
protein; b) covalently or non-covalently linking a pharmaceutically
active substance, label or pharmaceutically active substance and
label to and/or encapsulating a pharmaceutically active substance,
label or pharmaceutically active substance and label in an iron
binding protein; c) providing a CD45.sup.+ leukocyte cell; and d1)
incubating the CD45.sup.+ leukocyte cell in the presence of the
iron binding protein produced in step b) until the CD45.sup.+
leukocyte cell is at least partially loaded with the complex of the
iron binding protein and the a pharmaceutically active substance,
label or pharmaceutically active substance and label produced in
step b); and/or d2) incubating CD45.sup.+ leukocyte cell in the
presence of the label until the CD45.sup.+ leukocyte cell is at
least partially labelled with the label.
29. The isolated targeted delivery system of claim 1 for use as a
medicament or diagnostic.
30. A pharmaceutical composition comprising the isolated targeted
delivery system of claim 1 and a pharmaceutically acceptable
carrier and/or suitable excipient(s).
31. The isolated targeted delivery system of claim 1 for use in
preventing, treating or diagnosing a tumour, an inflammatory
disease or ischemic areas, or for prophylactic of therapeutic
vaccination, in particular to prevent or treat an infectious
disease or cancer.
Description
[0001] The present invention relates to an isolated cellular
targeted delivery system comprising a CD45.sup.+ leukocyte cell
comprising within said cell a complex of one or more iron binding
proteins and an active pharmaceutically active substance and/or
label as well as methods for producing such isolated cellular
targeted delivery system and uses of such system for prophylaxis,
therapy, diagnosis or theragnosis, in particular for prophylactic
or therapeutic vaccination, therapy of cancer, particularly
metastatic cancer or inflammatory diseases.
BACKGROUND OF THE INVENTION
[0002] Current imaging tools are capable of detecting large
metastases (larger than 0.5-1 cm in size). However, they rarely
detect the early spread of metastatic tumour cells. Human
metastases smaller than 0.5 cm are avascular so without proper
blood and oxygen supply. It means that the delivery of contrast
agents through the blood circulation for the purpose of labelling
these metastases and imaging them is not possible. The presence of
hypoxia is a common characteristic of micrometastases where hypoxic
fraction may be as high as 90% with little or no blood perfusion
(Li, et al. 2012, Journal of Solid Tumours, 2(2): 28-33). Thus,
severe hypoxia is considered as a general feature of
micro-metastases.
[0003] The targeting of one or more micrometastases hidden within a
large population of normal cells presents a unique challenge since
access to the micrometastases is impeded by several bio-barriers,
poor blood supply, further obstacles are presented by small size of
the micrometastases and their dispersion to organs.
[0004] For the same reason micrometastases are often refractive to
therapy. While the solid tumours from which the micrometastases
have originated often respond well to conventional therapy there is
often regrowth at the site of the primary tumour or at sites of
metastasis. This constitutes a serious problem in clinical oncology
(Muthana, et al. 2012, Cancer Res; 73(2); 490-495). It is related
to characteristics of the microenvironment of solid tumours that
limit drug penetration, thereby exposing the tumour to lower than
efficacious concentrations of drugs (Hobbs, et al. 1998, Proc Natl
Acad Sci USA: 4607-4612). This is caused by inadequate vasculature
resulting in: high heterogeneity of cancer cells, low oxygen
tension (hypoxia), low pH and low glucose concentration within the
mass (Kizaka-Kondoh, et al., 2003, Cancer Sci 94(12):1021-1028).
Additionally, rapid tumour cell proliferation in some areas might
outpace the rate of new blood vessel growth, promoting formation of
hypoxic area (Lewis and Murdoch, 2005, Am J Pathol 167(3):627-635).
This abnormal vessel architecture and, subsequently, their impaired
function resulting in tumour hypoxia is associated with a more
malignant phenotype and poor survival in patients suffering from
solid tumours and results in both treatment failure due to
decreased drug uptake and hypoxiainducible changes in cancer cells
(Sun, et al., 2012, Clin Cancer Res 18(3):758-770; Sullivan, et
al., 2008 Mol Cancer Ther 7(7):1961-1973; Kizaka-Kondoh, et al.,
2003, Cancer Sci 94(12):1021-1028). Moreover, chemotherapy or
radiotherapy causes additional formation of large areas of tumour
hypoxia thus making the treatment of tumour even more difficult.
The fact that the efficacy of anticancer therapy is limited by the
presence of hypoxic tumour cells has resulted in the introduction
of variety of therapeutic approaches aimed at overcoming this
problem.
[0005] The present inventors have discovered that CD45.sup.+
leukocyte cells, in particular activated macrophages, their
precursors monocytes, lymphocytes and granulocytes can uptake
pharmaceutically active substances, labels or pharmaceutically
active substances and labels complexed with one or more iron
binding proteins in vitro and deliver these complexes to or into
cells, preferably to or into tumour cells in vivo or other cells
undergoing stress (e.g. oxidative stress). Based on this
observation the present inventors have overcome one or more of the
above stated problems of the prior art. Thus, the targeted delivery
system of the present invention provides inta alia one or more of
the following advantages: (i) specific delivery of one or more
pharmaceutically active substances, labels or pharmaceutically
active substances and labels to tissues that attract above
mentioned CD45.sup.+ leukocytes, preferably into diseased cells,
(ii) protection of pharmaceutically active substances, labels or
pharmaceutically active substances and labels from inactivation in
the blood circulation or clearance from the body, (iii) delivery of
pharmaceutically active substances, labels or pharmaceutically
active substances and labels to, preferably into cells of poorly or
non-vascularized areas of disease, e.g. metastases, hypoxic areas
within larger tumours, rheumatic lesions, inflamed lymph nodes,
avascular wounds, skin, (iv) reduced toxicity of pharmaceutically
active substances, labels or pharmaceutically active substances and
labels, (v) delivery of pharmaceutically active substances, labels
or pharmaceutically active substances and labels with poor
pharmacokinetics, (vi) reduced side effects of the drugs due to
their targeted delivery, (vii) higher treatment efficacy with lower
doses of the drugs due to targeted delivery; and/or (viii) lower
risk of local tissue injury at the site of drug administration due
to administration of the drug linked with iron-binding protein,
which is loaded inside the CD45.sup.+ leukocyte; and/or (ix)
possibility to detect highly hypoxic small metastases; and/or (x)
early detection of inflammatory diseases.
SUMMARY OF THE INVENTION
[0006] In a first aspect the present invention relates to an
isolated targeted delivery system comprising a CD45.sup.+
monocyte-macrophage, CD45.sup.+ lymphocyte, in particular
CD45.sup.+ natural killer cells (NK cells), precursors of any of
these cell types, preferably MDSC, and/or CD45.sup.+ granulocyte,
("CD45.sup.+ leukocyte cell"), or their precursors, preferably
MDSC, comprising within said cell a complex of one or more iron
binding proteins and a pharmaceutically active substance, label or
pharmaceutically active substance and label.
[0007] In a second aspect the present invention relates to an
isolated targeted delivery label system comprising a CD45.sup.+
leukocyte cell, preferably capable of being labelled, comprising
one or more labels, preferably radiolabels or their conjugates and
combinations.
[0008] In a third aspect the present invention relates to a method
of preparation of the isolated targeted delivery system of claims 1
to 25 comprising steps of [0009] a) providing purified iron binding
protein; [0010] b) covalently or non-covalently linking a
pharmaceutically active substance, label or pharmaceutically active
substance and label to and/or encapsulating a pharmaceutically
active substance, label or pharmaceutically active substance and
label in an iron binding protein; [0011] c) providing a CD45.sup.+
leukocyte cell; and [0012] d1) incubating the CD45.sup.+ leukocyte
cell in the presence of the iron binding protein produced in step
b) until the CD45.sup.+ leukocyte cell is at least partially loaded
with the complex of the iron binding protein and the a
pharmaceutically active substance, label or pharmaceutically active
substance and label produced in step b); and/or [0013] d2)
incubating CD45.sup.+ leukocyte cell in the presence of the label
until the CD45.sup.+ leukocyte cell is at least partially labelled
with the label.
[0014] In a fourth aspect the present invention relates to an
isolated targeted delivery system of the first aspect or second
aspect of the invention or producible according to the method of
the third aspect of the invention for use as a medicament or
diagnostic.
[0015] In a fifth aspect the present invention relates to a
pharmaceutical or diagnostic composition comprising the isolated
targeted delivery system of the first or second aspect of the
present invention or producible according to the method of the
third aspect of the invention and a pharmaceutically acceptable
carrier and/or suitable excipient(s).
[0016] In a sixth aspect the present invention relates to an
isolated targeted delivery system of the first aspect or second
aspect of the present invention or producible according to the
method of the third aspect of the invention for use in preventing,
treating or diagnosing a disease characterized by hypoxic areas
within the diseases tissue and/or by areas of oxidative stress;
tumours, preferably solid tumours and/or its metastases, preferably
breast cancer, pancreatic cancer, bladder cancer, lung cancer,
colon cancer, or a tumour having hypoxic areas, an inflammatory
disease, inflamed tissue, preferably inflamed joints or arthritic
joints, inflamed lung, inflamed intestine or other inflamed tissue;
lymph nodes, preferably inflamed lymph nodes, or other
non-physiological lymph nodes that develop during disease
preferably but not only during infection, cancer, or autoimmune
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 also of
antigens to physiological or non-physiological lymph nodes in order
to vaccinate an individual or to induce immune memory.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0017] 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.
[0018] 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.
[0019] 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.
Definitions
[0020] 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, 2.sup.nd Edition, J. Sambrook et al. eds., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor 1989).
[0021] 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.
[0022] The term "targeted pharmaceutically active substance
delivery" refers to the delivery of a pharmaceutically active
substance to a patient which results in an increased concentration
of the pharmaceutically active substance 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 the 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 pharmaceutically active
substance 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 pharmaceutically active
substance delivery system of the present invention, preferably
after 2-24 hrs. More preferably, the concentration of the
pharmaceutically active substance 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
pharmaceutically active substance 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
pharmaceutically active substance 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
pharmaceutically active substance limits the potential deleterious
effects of a pharmaceutically active substance to the diseased
region of the body.
[0023] The term "targeted label delivery" or "targeted contrast
agent delivery" refers to the delivery of a label, in particular a
contrast agent to a patient or a person to be diagnosed which
results in an increased concentration of the label, in particular
the contrast agent 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 the 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 label, in particular a contrast agent 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
1-24 hrs. More preferably, the concentration of the label, in
particular the contrast agent 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 label, in particular the contrast agent administered to a
patient or a person to be diagnosed is delivered to the diseased
region of the body, preferably at least 10%, more preferably at
least 15%. The targeted delivery of the label, in particular the
contrast agent limits the potential deleterious effects of an
label, in particular the contrast agent to the diseased region of
the body.
[0024] The term "targeted theragnostic delivery" has a similar
meaning as set out above for "targeted pharmaceutically active
substance" and "targeted label delivery, however instead of only
delivering a pharmaceutical active substance or a label the complex
of the iron binding protein comprises in this embodiment both a
pharmaceutically active substance and a label, thereby allowing the
concomitant delivery of both a pharmaceutically active substance
and a label.
[0025] The term "targeted pharmaceutically active substance
delivery system" is used in the present application to refer to a
system that is capable of delivering a pharmaceutically active
substance to the targeted region, i.e. capable of targeted delivery
within the body of a patient, preferably to a diseased region.
[0026] The term "targeted label delivery system" is used in the
present application to refer to a system that is capable of
delivering a label to the targeted region, i.e. capable of targeted
delivery within the body of a patient, preferably to a diseased
region.
[0027] The term "targeted theragnostic delivery system" is used in
the present application to refer to a system that is capable of
delivering a complex of a pharmaceutically active substance and at
the same time a label to the targeted region, i.e. capable of
targeted delivery within the body of a patient, preferably to a
diseased region and thus allows simultaneous treatment and
diagnosis and/or treatment monitoring.
[0028] The term "targeted delivery system" is used to commonly
refer to "targeted pharmaceutically active substance delivery
system", "targeted label delivery system" and "targeted
theragnostic delivery system"
[0029] The term "pharmaceutically active substance" is used in the
context of the present invention to refer to a substance that
modifies or modulates cell activity, preferably a cell activity
that leads to a disease in a patient. The term encompasses both
substances which are already pharmaceutically active or which are
capable of being activated, i.e. a prodrug. Examples of such
pharmaceutically active substances include so called (i) "small
molecules", (ii) polynucleotides and (iii) peptide or proteins. 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, drugs that can be used comprise anti-cancer drugs,
pharmaceutically active radioactive isotopes or ferrihydrite.
[0030] 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.
[0031] 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 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),
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; U.S. Pat. No. 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-ribonucleotide (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 ribonucleases and,
therefore, possess greater stability.
[0032] The term "peptide" or "polypeptide" is 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
"peptide" 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.
[0033] 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.
[0034] 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').sub.2
fragment, a heavy chain antibody, a single-domain antibody (sdAb),
a single-chain fragment variable (scFv), a fragment variable (Fv),
a V.sub.H domain, a V.sub.L 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.
[0035] 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 (V.sub.H)
connected to a light-chain variable domain (V.sub.L) on the same
polypeptide chain (V.sub.H-V.sub.L, or V.sub.L-V.sub.H). 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).
[0036] 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 kDa and thus, are the smallest antibody
fragments capable of antigen recognition. Further examples include
nanobodies or nanoantibodies.
[0037] 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 to an antigen. Examples include inter alia the LACI-D1
(lipoprotein-associated coagulation inhibitor); affilins, e.g.
human-.gamma. B crystalline or human ubiquitin; cystatin; Sac7D
from Sulfolobus acidocaldarius; lipocalin and anticalins derived
from lipocalins; DARPins (designed ankyrin repeat domains); SH3
domain of Fyn; Kunits domain of protease inhibitors; monobodies,
e.g. the 10.sup.th type III domain of fibronectin; adnectins:
knottins (cysteine knot miniproteins); 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 composed from these. Common advantages over
antibodies are better solubility, tissue penetration, stability
towards heat and enzymes, and comparatively low production
costs.
[0038] 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.
[0039] 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.
[0040] An immunogenic polypeptide according to the present
invention is, preferably, derived from a pathogen selected from the
group consisting of viruses, bacteria and protozoa. In particular
embodiments. However, in an alternative embodiment of the present
invention the immunogenic polypeptide is a tumor antigen, i.e.
polypeptide or fragment of a polypeptide specifically expressed by
a cancer.
[0041] The term "sequence 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 (HMMER 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/Tools/clustalw/
or on http://www.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 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/clustalw2/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 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 Suppl 1:154-162) 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 N V: 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 O, 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.
[0042] The term "label" as used in the context of the present
invention refers 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 .sup.65Tb,
fluorescence emitting isotopes, such as .sup.18F, .sup.51Cr,
.sup.67Ga, .sup.68Ga, .sup.111In, .sup.99mTc, .sup.140La,
.sup.175Yb, .sup.153Sm, .sup.166Ho, .sup.88Y, .sup.89Zr, .sup.90Y,
.sup.149Pm, .sup.177Lu, .sup.47Sc, .sup.142Pr, .sup.159Gd,
.sup.212Bi, .sup.72As, .sup.72Se, .sup.97Ru, .sup.109Pd,
.sup.105Rh, .sup.101m15Rh, .sup.119Sb, .sup.128Ba, .sup.123I,
.sup.124I, .sup.131I, .sup.197Hg, .sup.211At, .sup.169Eu,
.sup.203Pb, .sup.212Pb, .sup.64Cu, .sup.67Cu, .sup.188Re,
.sup.186Re, .sup.198Au and .sup.199Ag as well as conjugates,
compounds comprising such isotopes and combinations of above with
proteins, peptides, small molecular inhibitors, antibodies or other
compounds, e.g. .sup.18F fluorodeoxyglucose (.sup.18F-FDG),
.sup.89Zr-oxide or .sup.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. IRDYE.TM.700DX) as
well as conjugates and combinations of these classes of dyes or
fluorescent .sup.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 number, i.e. iodine for computer
tomography (CT), microbubbles and carriers such as liposomes that
contain these contrast agents.
[0043] The term "leukocyte" 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).
[0044] The present inventors have observed that CD45.sup.+
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.sup.+ 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.sup.+ leukocyte cells" or
"CD45.sup.+ 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 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).
[0045] It is understood by the skilled person that CD45.sup.+
leukocyte cells as defined above unless of clonal origin are a
mixed population of different leukocytes which share the common
property of expressing CD45.sup.+ surface antigen. Accordingly,
subpopulations of cells within the diverse group of CD45.sup.+
leukocyte cells as defined above are characterized throughout the
specification by further functional and/or structural
characteristics. The term "CD45.sup.+" indicates that the majority
of cells within a population of cells or essentially all cells
express the CD45.sup.+ surface antigen. 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
leukocytes. Generally, a cell is considered to be positive, i.e. is
indicated to be "+", 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 "+", 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 1 to 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 Quantibrite.TM. 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). 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 CD45.sup.+ leukocytes. The term "low
expression" of a given protein refers to detectable expression of
that protein that is 30% or less of the highest expression level
found, i.e. number of copies of that protein per cell, in a
population of healthy CD45.sup.+ leukocytes. Preferably, the
"highest expression level" is determined as the average of the
highest expression levels found in healthy CD45.sup.+ 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 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 in vivo labeling 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, et. al., (2015) Methods in Molecular
Biology).
[0046] The term "differentiated monocyte" is used in the context of
the present invention to refer 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.sup.+ cell with
high expression of CX3CR1, low expression of CCR2 and Ly6C.sup.-
and (ii) CD11b.sup.+ cell with low expression of CX3CR1, high
expression of CCR2 and Ly6C.sup.+. After leaving the bone marrow,
mouse Ly6C.sup.+ monocytes differentiate into Ly6C.sup.- monocytes
in circulation. Similarly, in human monocyte differentiation, it is
accepted that CD14.sup.++ classical monocytes leave bone marrow and
differentiate into CD14.sup.++CD16.sup.+ intermediate monocytes and
sequentially to CD14.sup.+CD16.sup.++ 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 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.
[0047] Macrophages are tissue-resident professional phagocytes and
antigen-presenting cells (APC), which differentiate from
circulating peripheral blood monocytes (PBMs). The term "activated
macrophage" is used in the context of the present invention to
refer to any macrophage 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 (AAM). The classically activated M1-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, J Leukoc
Biol, 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 "M1 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 M1 or M2
macrophages.
[0048] 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.
[0049] The term "iron binding protein" as used refers to a protein
that non-covalently binds an iron ion. Examples of such proteins
comprise ferritin, haemoglobin, transferrin; and lactoferrin. Iron
binding proteins are bound by cellular surface receptors which
facilitate the internalization of these proteins into cells.
[0050] In a first aspect the present invention relates to an
isolated targeted delivery system comprising a CD45.sup.+ monocyte,
CD45.sup.+ monocyte-macrophage, CD45.sup.+ lymphocyte, CD45.sup.+
NK cell and/or CD45.sup.+ granulocyte (hereinafter referred to as
"CD45.sup.+ leukocyte cell") comprising within said cell a complex
of one or more iron binding proteins and a pharmaceutically active
substance, label or pharmaceutically active substance and label.
Preferably the CD45.sup.+ 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.
[0051] It has been surprisingly found by the present inventors that
CD45.sup.+ leukocyte cells, preferably activated macrophages,
preferably M1 macrophages, more preferably M2 macrophages acquire
pharmaceutically active substances, labels or pharmaceutically
active substances and labels and deliver the pharmaceutically
active substances, labels or pharmaceutically active substances and
labels in the amount sufficient (i) to be detected using imaging
systems to allowing them to be used to trace their location in the
body. In case of .sup.18F-FDG this was particularly preferable in
case of monocytes and activated monocytes, macrophages, activated
macrophages, preferably M1 macrophages and most preferably M2
macrophages. Administration of cells-loaded with a label
(.sup.18F-FDG, .sup.89Zr-oxide), preferably radioactive label can
visualize organ with accumulation of the labelled cells preferably
by positron emission tomography (PET) imaging, (ii) to elicit a
prophylactic or therapeutic effect or to allow a therapy and
diagnosis of a disease at the same time.
[0052] In a second aspect the present invention relates to an
isolated targeted label delivery system comprising a CD45.sup.+
leukocyte cell, preferably capable of being labelled with one or
more labels, preferably radiolabels or their conjugates and
combinations. It is a preferred embodiment of the second aspect of
the present invention that the cell is directly linked to or
labelled with label. The label may be within said cell, or on its
surface, preferably it is on the surface of the cell.
[0053] The following preferred embodiments in each case further
specify both the first and second aspect of the present
invention.
[0054] The ability of a given CD45.sup.+ leukocyte cell or cell
population to internalize iron binding proteins depends on the
expression of receptors involved in this internalization process.
Receptors that lead to internalization of ferritin comprise, e.g.
TfR, CXCR4, CD163, and TIM-2. The skilled person is well aware how
to measure the amount of uptake of an iron binding protein and
preferred method of measuring the uptake are described in the
Example Section below. The present inventors also noted that
subpopulations of CD45.sup.+ leukocyte cells have a certain
propensity to internalize one iron binding protein over another
iron binding protein and, thus can attain higher complex
concentrations and/or show less leakage of the complex from the
cells. Such CD45.sup.+ leukocyte subpopulations are described in
more detail below.
[0055] The phrase "complex of one or more iron binding proteins and
a pharmaceutically active substance, label or pharmaceutically
active substance and label" as used in the context of the present
invention refers to a composition in which one or more molecules of
the pharmaceutically active substance, label or pharmaceutically
active substance and label are covalently or non-covalently bound
to one or more iron binding proteins. The covalent or non-covalent
binding between the one or more iron binding proteins and the one
more pharmaceutically active substance, label or pharmaceutically
active substance and label can be direct or indirect. In the latter
case the pharmaceutically active substance, label or
pharmaceutically active substance and label is linked to the iron
binding protein via a linker or spacer. Linker or spacers are known
to the skilled artisan, such as polyalanine, polyglycin,
carbohydrates, (CH.sub.2)n groups or polypeptide linkers. The
skilled artisan will, thus, be able to select the respective
suitable linker(s) or spacer(s) depending on the respective
application. If the iron binding proteins form cages like, e.g.
ferritin, than the term "complex" also encompasses the enclosure of
pharmaceutically active substances, labels or pharmaceutically
active substances and labels within the cage even in the absence of
a covalent or non-covalent bond between the protein(s) and the
active compound(s). The formation of the complex allows the
transport of the pharmaceutically active substance, label or
pharmaceutically active substance and label into the cell when the
cell is internalizing the iron binding protein. Thus, it is
preferred that the pharmaceutically active substances, labels or
pharmaceutically active substances and labels 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. 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
pharmaceutically active substance, label or pharmaceutically active
substance and label 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 pharmaceutically
active substance, label or pharmaceutically active substance and
label to the CD45.sup.+ leukocyte cell. If pharmaceutically active
substances, labels or pharmaceutically active substances and labels
are covalently coupled to the iron binding proteins such coupling
is preferably through amino acids residues known to be located in
surface areas that are not involved in binding to the cellular
receptors required for cellular uptake of the iron binding
proteins. Iron binding proteins used in the context of the present
invention can form stable non-covalently bound complexes with a
wide variety of pharmaceutically active substances, labels or
pharmaceutically active substances and labels. If the
pharmaceutically active substance or label 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 iron binding protein peptide fusion
protein.
[0056] The CD45.sup.+ leukocyte cells 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 of the
present invention and that the leukocyte cell type used for a given
patient is MHC matched to the patient. In these two preferred
embodiments the CD45.sup.+ leukocyte cell is a primary cell or
derived by a low number of differentiation steps from a primary
cell. Alternatively, the CD45.sup.+ leukocyte cell may be from an
immortalized but preferably non-transformed CD45.sup.+ leukocyte
cell line. Thus the blood used for CD45.sup.+ leukocyte cells, i.e.
CD45.sup.+ monocyte, CD45.sup.+ monocyte-macrophage, CD45.sup.+
granulocyte, or CD45.sup.+ lymphocyte, in particular CD45.sup.+ NK
cell, isolation 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.
[0057] The present inventors noted that a subpopulation of
CD45.sup.+ leukocyte cells, which are producible from a CD34.sup.+
hematopoietic precursor cell are particular suitable for target
specific delivery of the pharmaceutically active substance, label
or pharmaceutically active substance and label. Accordingly, it is
preferred that the leukocytes used to produce the target delivery
system are derived from CD34.sup.+ hematopoietic precursor cells.
The skilled person is well aware how to select CD34.sup.+
hematopoietic precursor cells and how to differentiate such cells
into leukocytes.
[0058] As indicated above, the term "CD45.sup.+ leukocyte cell" is
used throughout this specification to refer to a CD45.sup.+
monocyte, CD45.sup.+ monocyte-macrophage, CD45.sup.+ lymphocyte
and/or CD45.sup.+. 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, 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.
[0059] The present inventors have recognized that subpopulations of
CD45.sup.+ leukocyte cells have particular advantageous properties
including among others efficiency and/amount of complex uptake in
general, ability to retain the complex within the cell, i.e. to
avoid leakage and of target release of the pharmaceutically active
substance, label or pharmaceutically active substance and label,
efficiency of uptake of a particular iron binding protein and/or
targeting to particular tissues or cells and, thus suitability to
treat or ameliorate a particular disease. The present inventors
have, e.g. observed that CD45.sup.+ leukocyte cells, which express
one or more of the following antigens: CD204, CD206, CD200R, CCR2
have a preference for ferritin uptake over the uptake of other iron
binding proteins. Thus, if the iron binding protein in the complex
is ferritin it is preferred to select CD45.sup.+ leukocyte cells
that express one or more of the following antigens: CD204, CD206,
CD200R, CCR2. Accordingly, in a preferred embodiment of the present
invention [0060] (i) the monocyte is a CD11b.sup.+ monocyte,
preferably selected from the group consisting of a CD11b.sup.+
CD14.sup.+ monocyte, a CD11b.sup.+ CD16.sup.+ monocyte, a
CD11b.sup.+ CD14.sup.+ CD16.sup.+ monocyte, a CD11b.sup.+
CD14.sup.+ MHCII.sup.+ monocyte, a CD11b.sup.+ CD14.sup.+
CD115.sup.+ monocyte, CD11b.sup.+ CD114.sup.+ monocyte, CD11b.sup.+
CD116.sup.+ monocyte, CD11b.sup.+ CCR1.sup.+ monocyte, CD11b.sup.+
CCR2.sup.+ monocyte, CD11b.sup.+ CX3CR.sup.+ monocyte, CD11b.sup.+
CXR4.sup.+ monocyte, CD11b.sup.+ CXR6.sup.+ monocyte and a
CD11b.sup.+ CD14.sup.+ CD33.sup.+ 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, IIRF4, and/or TCF4, and more preferably is not a
dendritic cell; [0061] (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 CD11b.sup.+ macrophage, more preferably a CD11b.sup.+
CD16.sup.+ macrophage, CD11b.sup.+ CD32.sup.+ macrophage,
CD11b.sup.+ CD64.sup.+ macrophage, CD11b.sup.+ CD68.sup.+
macrophage, preferably a CD11b.sup.+ CD86.sup.+ M1 macrophage,
preferably producing inducible nitric oxide synthetase (iNOS)
and/or secreting interleukin 12 (IL-12) or preferably CD11b.sup.+
CCR2.sup.+ M2 macrophage, CD11b.sup.+ CD204.sup.+ M2 macrophage,
CD11b.sup.+ CD206.sup.+ M2 macrophage, CD11b.sup.+ CD204.sup.+
CD206.sup.+ M2 macrophage, CD11b.sup.+ Major Histocompatibility
Complex II.sup.+ (MHCII.sup.+) (low or hi expression) M2
macrophage, CD11b.sup.+ CD200R.sup.+ M2 macrophage, CD11b.sup.+
CD163.sup.+ M2 macrophage or activated macrophage producing and/or
secreting Arginase-1 and/or interleukin 10 (IL10); preferably the
differentiated monocyte is not a foam cell expressing Lectin-like
oxidized low-density lipoprotein receptor-1 (Lox1.sup.+), C--X--C
chemokine receptor type 7 (CXCR7.sup.+) and Nuclear factor
(erythroid-derived 2)-like 2 (NRF2.sup.+). 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;
[0062] (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 (CD115), granulocyte
colony stimulating factor Receptor (CD114), and
granulocyte-macrophage colony stimulating factor Receptor
(consisting of CD116 and CD131); monocytes of these characteristics
are particular suitable to treat inflammatory conditions and
cancer; [0063] (iv) the lymphocyte is selected from the group
consisting of a CD3.sup.+ and CD4.sup.+ or CD8.sup.+ T lymphocyte,
or a CD19.sup.+, CD20.sup.+, CD21.sup.+, CD19.sup.+ CD20.sup.+,
CD19.sup.+ CD21.sup.+, CD20.sup.+ CD21.sup.+, or CD19.sup.+
CD20.sup.+ CD21.sup.+ B lymphocyte; or a natural killer (NK) cell,
preferably the NK cell is selected from the group consisting of
CD56.sup.+ and without CD3 expression, or CD16.sup.+ CD56.sup.+,
CD56.sup.+ CD94.sup.+, CD56.sup.+ CD158a.sup.+, CD56.sup.+
CD158f.sup.+, CD56.sup.+CD314.sup.+, CD56.sup.+CD335.sup.+ cell; or
[0064] (v) the granulocyte is selected from the group consisting of
a neutrophil, preferably a CD66b.sup.+ neutrophil, an eosinophil
and a basophil, preferably a CD193.sup.+ eosinophil.
[0065] In a preferred embodiment of the targeted delivery system of
the present invention the activated macrophage: [0066] (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 [0067] (a) with at least one M1
inducer, [0068] (b) with at least one M2 inducer, [0069] (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-.beta.
(TGF-.beta.), IL-1R, CC-chemokine ligand 2 (CCL-2), IL-6,
Macrophage colony-stimulating factor (M-CSF), peroxisome
proliferator-activated receptor .gamma. (PPAR.gamma.) agonist,
leukocyte inhibitory factor (LIF), adenosine, helminth and fungal
infection, lipopolysaccharide (LPS), interferon .gamma.
(INF-.gamma.), 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, [0070] (ii) is
characterized by expression of at least one of following antigens:
CD64, CD86, CD16, CD32, high expression of MCHII, and/or production
of iNOS and/or IL-12; [0071] (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 .gamma. (INF-.gamma.), viral
infection and/or bacterial infection; [0072] (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 MHCII;
activated macrophages having these properties are particularly
suitable for complexes comprising ferritin as the iron binding
protein; [0073] (v) has the ability to phagocytose; and/or [0074]
(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.
[0075] In a preferred embodiment of the targeted delivery system of
the present invention the M1 inducer for differentiating
macrophages into M1 macrophages is selected from the group
consisting of lipopolysaccharide (LPS), interferon .gamma.
(INF-.gamma.), 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 gamma-globulin,
glucocorticosteroid, tumour growth factor-0 (TGF-.beta.), IL-1R,
CC-chemokine ligand 2 (CCL-2), IL-6, Macrophage colony-stimulating
factor (M-CSF), peroxisome proliferator-activated receptor .gamma.
(PPAR.gamma.) agonist, leukocyte inhibitory factor (LIF),
adenosine, helminth and fungal infection.
[0076] It has been surprisingly found by the present inventors that
both complex loaded M1 macrophages and M2 macrophages are suitable
for targeted active agent delivery into hypoxic tissues, preferably
a tumour or its metastasis. In general we observed that 3 to 5% of
the administered M1 macrophages localized at the tumour site while
about 35% of the M2 macrophages showed tumour specific targeting.
However, this general advantage of M2 macrophages were offset when
using a complex comprising haemoglobin and drug, since
significantly larger amounts of this complex could be loaded into
M1 macrophages than into M2 macrophages. Generally this specific
tropisms makes M2 macrophages more suitable for treating tumour and
diseases characterized by hypoxic tissue.
[0077] The present inventors also found CCL2-activated macrophages
(bone marrow derived) are particularly suitable to target lymph
nodes, preferably inflamed lymph nodes. Thus, the use of such
macrophages is preferred in all uses in which the targeting of
lymph nodes is desired, in particular for prophylactic or
therapeutic vaccination as well as for diagnosis of inflamed lymph
nodes. Accordingly, the loading of CCL2-activated macrophages with
complexes of iron binding proteins and antigens is preferred.
[0078] In a preferred embodiment of the targeted delivery system of
the present invention the monocyte-macrophage: [0079] (i) is
producible from a CD34.sup.+ hematopoietic precursor cell; [0080]
(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; [0081] (iii) is characterized by expression
of at least one of the following antigens: TfR, CD163, TIM-2, CD14,
CD16, CD33, and/or CD115; [0082] (iv) is characterized by
expression of at least one of the following antigens: TfR, CD163,
TIM-2, CXCR4, CD14, and/or CD16; and/or [0083] (v) has the ability
to phagocytose; and/or [0084] (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.
[0085] 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-.gamma. 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, Glucocorticosteroids,
TGF-.beta., IL-1R, CCL-2, IL-6, M-CSF, PPAR.gamma. agonist,
Leukocyte inhibitory factor (LIF), cancer-conditioned medium,
cancer cells, adenosine and helminth or fungal infection.
[0086] It has been surprisingly found by the present inventors that
monocytes are suitable for targeted active agent delivery into
hypoxic tissues, preferably the tumour or its metastasis while
monocytes treated with M2 activators are more suitable for targeted
active agent delivery into hypoxic tissues, preferably the tumour
or its metastasis. This specific tropisms make monocytes treated
with M2 activators more suitable to targeting tumour and hypoxic
sites, e.g. sites of inflammation.
[0087] In a preferred embodiment of the targeted delivery system of
the present invention the lymphocyte: [0088] (i) is obtainable from
blood, spleen, or bone marrow or is producible from a CD34.sup.+
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; [0089] (ii) is an immunologically
competent lymphocyte; [0090] (iii) expresses antigen specific T
cell receptors; and/or [0091] (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 [0092] (i) is obtainable from
blood, spleen or bone marrow or producible from a CD34.sup.+
precursor cell; and/or [0093] (ii) is characterized by the lack of
CD3 expression and expression of at least one of the following
CD56.sup.+ and/or CD94.sup.+, CD158a.sup.+ CD158f.sub.+ CD314.sup.+
CD335.sup.+.
[0094] In a preferred embodiment of the targeted delivery system of
the present invention the granulocyte: [0095] (i) is obtainable
from blood, spleen or bone marrow or producible from a CD34.sup.+
precursor cell as described, e.g. in Kuhs et al. 2015, Curr Protoc
Immunol 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; [0096]
(ii) is characterized by expression of at least one of the
following CD66b and/or CD193; [0097] (iii) is a polymorphonuclear
leukocyte characterized by the presence of granules in its
cytoplasm; and/or [0098] (iii) is characterized by expression of at
least one of the following: TfR, CD163, TIM-2, and/or CXCR4.
[0099] In a preferred embodiment of the targeted delivery system of
the present invention the iron binding protein is selected from the
group consisting of ferritin, preferably heavy (H) type ferritin,
light (L) ferritin and/or mitochondrial ferritin; haemoglobin,
preferably haemoglobin A, haemoglobin AS, haemoglobin SC,
haemoglobin C, haemoglobin D, haemoglobin E, haemoglobin F,
haemoglobin H; haemoglobin-haptoglobin complex, haemopexin,
transferrin; and lactoferrin. The terms ferritin; haemoglobin,
preferably haemoglobin A, haemoglobin AS, haemoglobin SC,
haemoglobin C, haemoglobin D, haemoglobin E, haemoglobin F,
haemoglobin H; haemoglobin-haptoglobin complex, hemopexin,
transferrin; and lactoferrin encompass structural variants of the
naturally occurring proteins and, thus relates to proteins that
have at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, more preferably at least 95% more preferably at least 100% of
the ability of the respective wild-type protein to bind iron
ion(s). The iron binding proteins used in the context of the
present invention are preferably of mammalian, more preferably
mouse, rat, dog, ape, in particular, chimpanzee, or human, most
preferably of human origin. Consensus sequences of the preferred
iron binding proteins used in the context of the present invention
are shown in FIG. 1 below. Preferred structural variants are based
on the sequences indicated in FIG. 1. The residues marked with X
vary among different mammalian ferritins, transferrins, and
haemoglobins. The alteration of these residues is not crucial for
the ability of the proteins to bind to iron ions. Accordingly, it
is preferred that amino acid mutations or deletions effect one or
more of the residues marked with an X.
[0100] Plasma proteins have always been privileged carriers for the
delivery of active pharrrra ingredients in cancer therapy. Thus,
albumin, the most abundant plasma protein, is currently used in
therapeutic protocols for the delivery of taxane molecules and
doxorubicin derivatives (Larsen M T et al. 2016, Mol Cell Ther 27;
4:3).
[0101] Human transferrin and ferritin proteins have been considered
as effective carriers for the delivery of small molecules or
toxin-conjugates to specifically target cancer cells. To date; in
spite of considerable efforts, no successful transferrin or
ferritin drug complexes have however reached the clinic (Luck A N
et al. 2013, Adv Drug Deliv Rev 65(8):1012-9).
[0102] Ferritin has a cage architecture and capability of
iron-binding which could be used to encapsulate pharmaceutically
active substances and/or labels inside its cavity. Ferritins are
not abundant in plasma, but can be readily produced in high yield
as recombinant proteins in common protein expression vectors such
as Escherichia coli cells. Ferritins H- or L-chains are encoded as
small protein monomer (21 kDa and 19 KDa for H and L chains,
respectively) capable of a 24-mer assembly into a cage-like
structure, delimiting a 8 nm diameter cavity. The present inventors
noted in the context of working on the present invention that
H-ferritin homopolymers, represent a preferred protein construct in
order to specifically deliver encapsulated drugs to CD45.sup.+
leukocyte cells expressing TfR. Furthermore H-ferritin targets
complex pharmaceutically active substances, labels or
pharmaceutically active substances and labels to the cell nucleus
(and therefore directly delivers DNA-binding proteins into the
nucleus.
[0103] Purified transferrin can be efficiently conjugated to
various molecules including anticancer drugs through covalent
linkers that are appropriately released inside the cells (Beyer U
et al. 1998, J Med Chem 41(15):2701-2708). In case of transferrin,
only lysine groups on the protein surface are ready available for
covalent attachment.
[0104] Haemoglobin has been considered in the past as a possible
drug carrier, due to its versatility in chemical conjugation with
drugs, its abundance and relative stability in the blood
(Somatogen, 1993, WO1993008842 A1). Nevertheless, the lack of
receptor targeting properties did not foster biomedical
applications other than blood substitutes or antisickling agent. As
a matter of fact, Hb can only be recognized by CD163
(haptoglobin/haemoglobin receptor) epitopes on the leukocytes,
especially monocyte-macrophage origin. The CD45.sup.+ leukocyte, in
particular macrophage based protein delivery, described in this
application moved haemoglobin center stage as a target specific
carrier of pharmaceutically active substances and/or labels.
Haemoglobin can be readily covalently linked to appropriate
pharmaceutically active substances and/or labels, host hydrophobic
pharmaceutically active substances and/or labels within the heme
binding pocket or even transport small molecules, e.g. cytotoxic
molecules linked to the heme iron. Hb can be easily modified by
selective attachment of the appropriate drug conjugate to the
beta93 cysteine residue, the only titratable cystein on the protein
surface. Maleimido functionalized drugs, such as the tubuline
inhibitor MonomethylAuristatin (MMAE) or the DNA crosslinking drug
Pyrrolobenzodiazepine dimer (PBD) are most notable examples of
extremely potent cytotoxics that can be readily and specifically
attached to the relevant cys beta93 residue.
[0105] Alternatively, lysine residues on the Hb surface (at least
10 titratable lysine residues per Hb tetramer) may be easily
amenable to drug conjugation through cleavable succinimide linkers.
Haemoglobin also offers a unique capability of releasing non
covalently bound heme group at acidic pH values. Apo-haemoglobin
thus obtained is capable of hosting several hydrophobic molecules
within the empty heme pocket, as shown in the case of paclitaxel
(Meng Z et al. 2015 J Pharm Sci 104(3):1045-55) or for labels with
fluorescent properties (e.g. chlorine e6, hyperycin, phtalocyanine
derivatives) (Bong J et al. J Photochem Photobiol B 2014,
140:163-172).
[0106] Whatever the conjugation/adsorption/binding method,
haemoglobin (Fib), transferrin (Tf) and ferritins were shown by the
present inventors to be privileged carriers of pharmaceutically
active substances and labels, once loaded into appropriate cell
systems with tumour targeting properties, e.g. activated
macrophages. The easy, fast, cheap and safe purification procedure
of these protein also provide a tremendous added value.
[0107] Based on sequences of mammalian H-type ferritins, L-type
ferritins, haemoglobin alpha chains, haemoglobin beta chains and
transferrins a consensus sequence was determined for each of these
proteins. These are shown in FIG. 1 in SEQ ID NO: 2, 7, 9, 14, 16,
20, and 25, respectively). On this basis but also on the basis of
deletion and structural analysis disclosed in the prior art a
minimal fragment was determined for H-type ferritins, L-type
ferritins, haemoglobin alpha chains, and haemoglobin beta chains
sufficient for uptake by CD45.sup.+ leukocytes. These are shown in
SEQ ID NO: 1, 3, 5 (H-type ferritin); 8, 10, 12 (L-type ferritin),
15 and 17 (haemoglobin alpha chain) and 19 and 21 (haemoglobin beta
chain. Transferrin comprises a N-terminally located domain and a
C-terminally located domain that are necessary for binding iron and
uptake by CD45.sup.+ leukocytes, if comprised in one polypeptide
and positioned between 100 to 450 amino acids apart, preferably
between 150 to 400, more preferably between 200 to 350 amino acids
and more preferably 250 to 320 amino acids apart. The N-terminal
domain comprises amino acids 1 to 82 of full length consensus
transferrin (SEQ ID NO: 25) or full length human transferrin (SEQ
ID NO: 28). The C-terminal domain comprises amino acids 396 to 510
of full length consensus transferrin (SEQ ID NO: 25) or full length
human transferrin (SEQ ID NO: 28). In each case an X is indicated
in the consensus sequence it independently stands for any amino
acid and characterizes an amino acid not or only poorly conserved
among mammalian H-type ferritins, which can be mutated without or
little detriment to the iron binding properties of the respective
iron binding protein. It is preferred that X in each case takes on
the meaning of the amino acid of the respective human iron binding
protein aligning with X. This information can be taken, e.g. from
FIG. 1, which shows alignments of the consensus sequences with
human an in some instances mouse proteins.
[0108] Preferred H-type ferritins comprise or consist of the amino
acid sequence indicated in SEQ ID NO: 1 and variants thereof having
at least 70% amino acid identity, more preferably at least 75%
amino acid identity, more preferably at least 80% amino acid
identity, more preferably at least 85% amino acid identity, more
preferably at least 90% amino acid identity, more preferably at
least 95% amino acid identity and in each case at least 70% of the
ability of a H-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 1 to be taken up by CD45.sup.+ leukocytes,
preferably M2 leukocytes. Within SEQ ID NO: 1 X at position 5 may
be present or absent, if present it means any amino acid,
preferably Ile, X at position 6 means any amino acid, preferably
Asn, X at position 14 means any amino acid, preferably Tyr, X at
position 24 means any amino acid, preferably Tyr or Cys, X at
position 66 means any amino acid, preferably Phe, X at position 68
means any amino acid, preferably Gln, X at position 75 means any
amino acid, preferably Arg or Cys, X at position 90 means any amino
acid, preferably His, X at position 94 means any amino acid,
preferably Ser or Asn, X at position 120 may be present or absent,
if present it means any amino acid, preferably His or Tyr, more
preferably His, X at position 123 means any amino acid, preferably
Asn or Ser, more preferably Asn, X at position 128 means any amino
acid, preferably Ala or Ser, more preferably Ala.
[0109] In a preferred embodiment the H-type ferritin comprises or
consists of murine ferritin according to SEQ ID NO: 3. Accordingly,
preferred structural variants have at least 70% amino acid
identity, more preferably at least 75% amino acid identity, more
preferably at least 80% amino acid identity, more preferably at
least 85% amino acid identity, more preferably at least 90% amino
acid identity, more preferably at least 95% amino acid identity and
in each case at least 70% of the ability of a H-type ferritin
consisting of the amino acid sequence according to SEQ ID NO: 3 to
be taken up by CD45.sup.+ leukocytes, preferably M2
macrophages.
[0110] In a preferred embodiment the H-type ferritin comprises or
consists of human ferritin according to SEQ ID NO: 5. Accordingly,
preferred structural variants have at least 70% amino acid
identity, more preferably at least 75% amino acid identity, more
preferably at least 80% amino acid identity, more preferably at
least 85% amino acid identity, more preferably at least 90% amino
acid identity, more preferably at least 95% amino acid identity and
in each case at least 70% of the ability of a H-type ferritin
consisting of the amino acid sequence according to SEQ ID NO: 5 to
be taken up by CD45.sup.+ leukocytes, preferably M2
macrophages.
[0111] In a preferred embodiment the H-type ferritin comprises or
consists of a mammalian consensus sequence derived from aligning
full length H-type ferritins according to SEQ ID NO: 2 or 7, with 2
being preferred. Accordingly, preferred structural variants have at
least 70% amino acid identity, more preferably at least 75% amino
acid identity, more preferably at least 80% amino acid identity,
more preferably at least 85% amino acid identity, more preferably
at least 90% amino acid identity, more preferably at least 95%
amino acid identity and in each case at least 70% of the ability of
a H-type ferritin consisting of the amino acid sequence according
to SEQ ID NO: 2 or 7, with 2 being preferred to be taken up by
CD45.sup.+ leukocytes, preferably M2 macrophages. In SEQ ID NO: 2 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 acid, preferably Glu, X at position 183 is absent
or any naturally occurring amino acid, preferably Ser. In SEQ ID
NO: 7 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 29 can be any naturally occurring amino acid,
preferably Tyr, X at position 81 can be any naturally occurring
amino acid, preferably Phe, X at position 83 can be any naturally
occurring amino acid, preferably Gln, X at position 105 can be any
naturally occurring amino acid, preferably His, X at position 144
can be any naturally occurring amino acid, preferably Ala or Ser,
more preferably Ala, X at position 180 is absent or any naturally
occurring amino acid, preferably Asn, X at position 181 is absent
or any naturally occurring amino acid, preferably Glu, X at
position 182 is absent or any naturally occurring amino acid,
preferably Ser.
[0112] In a preferred embodiment the H-type ferritin comprises or
consists of murine full length ferritin according to SEQ ID NO: 4
being preferred. Accordingly, preferred structural variants have at
least 70% amino acid identity, more preferably at least 75% amino
acid identity, more preferably at least 80% amino acid identity,
more preferably at least 85% amino acid identity, more preferably
at least 90% amino acid identity, more preferably at least 95%
amino acid identity and in each case at least 70% of the ability of
a murine H-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 4 to be taken up by CD45.sup.+ leukocytes,
preferably M2 macrophages.
[0113] In a preferred embodiment the H-type ferritin comprises or
consists of human full length ferritin according to SEQ ID NO: 6
being preferred. Accordingly, preferred structural variants have at
least 70% amino acid identity, more preferably at least 75% amino
acid identity, more preferably at least 80% amino acid identity,
more preferably at least 85% amino acid identity, more preferably
at least 90% amino acid identity, more preferably at least 95%
amino acid identity and in each case at least 70% of the ability of
a human H-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 6 to be taken up by CD45.sup.+ leukocytes,
preferably M2 macrophages.
[0114] Preferred L-type ferritins comprise or consist of the amino
acid sequence indicated in SEQ ID NO: 8 and variants thereof having
at least 70% amino acid identity, more preferably at least 75%
amino acid identity, more preferably at least 80% amino acid
identity, more preferably at least 85% amino acid identity, more
preferably at least 90% amino acid identity, more preferably at
least 95% amino acid identity and in each case at least 70% of the
ability of a L-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 8 to be taken up by CD45.sup.+ leukocytes,
preferably M2 leukocytes. In SEQ ID NO: 8 X at position at position
5 can be any naturally occurring amino acid, preferably Asp or Glu,
more preferably Asp, X at position 12 can be any naturally
occurring amino acid, preferably Arg or Ser, more preferably Ser, X
at position 17 can be any naturally occurring amino acid,
preferably Ser or Arg, more preferably Ser, X at position 19 can be
any naturally occurring amino acid, preferably Arg or Gln, more
preferably Gln, X at position 29 can be any naturally occurring
amino acid, preferably Phe, X at position 30 can be any naturally
occurring amino acid, preferably Tyr or Phe, more preferably Tyr, X
at position 42 can be any naturally occurring amino acid,
preferably Ser or Gly, more preferably Ser, X at position 56 can be
any naturally occurring amino acid, preferably Ala or Tyr, more
preferably Tyr, X at position 61 can be any naturally occurring
amino acid, preferably Glu or Lys, more preferably Lys, X at
position 62 can be any naturally occurring amino acid, preferably
Met or Phe, more preferably Met, X at position 65 can be any
naturally occurring amino acid, preferably Asp or Gln, more
preferably Gln, X at position 75 can be any naturally occurring
amino acid, preferably Ile or Val, more preferably Ile, X at
position 76 can be any naturally occurring amino acid, preferably
Lys or Gln, more preferably Lys, X at position 79 can be any
naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala, X at position 80 can be any naturally occurring
amino acid, preferably Glu or Gln, more preferably Gln, X at
position 87 can be any naturally occurring amino acid, preferably
Pro or Gln, more preferably Pro, X at position 88 can be any
naturally occurring amino acid, preferably Glu or Asp, more
preferably Asp, X at position 91 can be any naturally occurring
amino acid, preferably Glu or Lys, more preferably Lys, X at
position 94 can be any naturally occurring amino acid, preferably
Met or Leu, more preferably Leu, X at position 96 can be any
naturally occurring amino acid, preferably Met or Leu, more
preferably Met, X at position 99 can be any naturally occurring
amino acid, preferably Lys or Asn, preferably Lys, X at position
115 can be any naturally occurring amino acid, preferably Thr or
Ala, more preferably Thr, X at position 119 can be any naturally
occurring amino acid, preferably Leu, X at position 125 can be any
naturally occurring amino acid, preferably Ser or Thr, more
preferably Thr, X at position 127 can be any naturally occurring
amino acid, preferably Tyr or Phe, more preferably Phe, X at
position 130 can be any naturally occurring amino acid, preferably
Lys or Glu, more preferably Glu, X at position 140 can be any
naturally occurring amino acid, preferably Asp or Asn, more
preferably Asp, X at position 146 can be any naturally occurring
amino acid, preferably Arg or His, more preferably His, and X at
position 148 can be any naturally occurring amino acid, preferably
Leu or Val, more preferably Leu.
[0115] In a preferred embodiment the L-type ferritin comprises or
consists of murine L-type ferritin according to SEQ ID NO: 10.
Accordingly, preferred structural variants have at least 70% amino
acid identity, more preferably at least 75% amino acid identity,
more preferably at least 80% amino acid identity, more preferably
at least 85% amino acid identity, more preferably at least 90%
amino acid identity, more preferably at least 95% amino acid
identity and in each case at least 70% of the ability of a L-type
ferritin consisting of the amino acid sequence according to SEQ ID
NO: 10 to be taken up by CD45.sup.+ leukocytes, preferably M2
macrophages.
[0116] In a preferred embodiment the L-type ferritin comprises or
consists of human ferritin according to SEQ ID NO: 12. Accordingly,
preferred structural variants have at least 70% amino acid
identity, more preferably at least 75% amino acid identity, more
preferably at least 80% amino acid identity, more preferably at
least 85% amino acid identity, more preferably at least 90% amino
acid identity, more preferably at least 95% amino acid identity and
in each case at least 70% of the ability of a L-type ferritin
consisting of the amino acid sequence according to SEQ ID NO: 12 to
be taken up by CD45.sup.+ leukocytes, preferably M2
macrophages.
[0117] In a preferred embodiment the L-type ferritin comprises or
consists of a mammalian consensus sequence derived from aligning
full length H-type ferritins according to SEQ ID NO: 9 or 14, with
9 being preferred. Accordingly, preferred structural variants have
at least 70% amino acid identity, more preferably at least 75%
amino acid identity, more preferably at least 80% amino acid
identity, more preferably at least 85% amino acid identity, more
preferably at least 90% amino acid identity, more preferably at
least 95% amino acid identity and in each case at least 70% of the
ability of a L-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 9 or 14, with 9 being preferred to be taken
up by CD45.sup.+ leukocytes, preferably M2 macrophages. In SEQ ID
NO: 9 X at position 12 X at position 12 can be any naturally
occurring amino acid, preferably Asp or Glu, more preferably Asp, X
at position 19 can be any naturally occurring amino acid,
preferably Ser or Arg, more preferably Ser, X at position 24 can be
any naturally occurring amino acid, preferably Ser or Arg, more
preferably Ser, X at position 26 can be any naturally occurring
amino acid, preferably Arg or Gln, more preferably Gln, X at
position 36 can be any naturally occurring amino acid, preferably
Phe, X at position 37 can be any naturally occurring amino acid,
preferably Tyr or Phe, more preferably Tyr, X at position 47 can be
any naturally occurring amino acid, preferably Ser or Gly, more
preferably Ser, X at position 63 can be any naturally occurring
amino acid, preferably Ala or Tyr, more preferably Tyr, X at
position 68 can be any naturally occurring amino acid, preferably
Glu or Lys, more preferably Lys, X at position 69 can be any
naturally occurring amino acid, preferably Met or Phe, more
preferably Met, X at position 72 can be any naturally occurring
amino acid, preferably Asp or Gln, more preferably Gln, X at
position 82 can be any naturally occurring amino acid, preferably
Ile or Val, more preferably Ile, X at position 83 can be any
naturally occurring amino acid, preferably Lys or Gln, more
preferably Lys, X at position 86 can be any naturally occurring
amino acid, preferably Ala or Ser, more preferably Ala, X at
position 87 can be any naturally occurring amino acid, preferably
Glu or Gln, more preferably Gln, X at position 94 can be any
naturally occurring amino acid, preferably Pro or Gln, more
preferably Pro, X at position 95 can be any naturally occurring
amino acid, preferably Glu or Asp, more preferably Asp, X at
position 98 can be any naturally occurring amino acid, preferably
Glu or Lys, more preferably Lys, X at position 101 can be any
naturally occurring amino acid, preferably Met or Leu, more
preferably Leu, X at position 103 can be any naturally occurring
amino acid, preferably Met or Leu, more preferably Met, X at
position 106 can be any naturally occurring amino acid, preferably
Lys or Asn, preferably Lys, X can be any naturally occurring amino
acid, X at position 126 can be any naturally occurring amino acid,
preferably Leu, X at position 132 can be any naturally occurring
amino acid, preferably Ser or Thr, more preferably Thr, X at
position 134 can be any naturally occurring amino acid, preferably
Tyr or Phe, more preferably Phe, X at position 137 can be any
naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu, X at position 147 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp, X at
position 153 can be any naturally occurring amino acid, preferably
Arg or His, more preferably His, X at position 155 can be any
naturally occurring amino acid, preferably Leu or Val, more
preferably Leu, X at position 161 can be absent or any naturally
occurring amino acid, preferably Ala, X at position 163 can be
absent or any naturally occurring amino acid, preferably Thr, X at
position 166 can be absent or any naturally occurring amino acid,
preferably Pro, and X at position 168 can be any naturally
occurring amino acid, preferably Gly or Ala, more preferably Ala.
In SEQ ID NO: 14 X at position 36 can be any naturally occurring
amino acid, preferably Phe, X at position 37 can be any naturally
occurring amino acid, preferably Tyr or Phe, more preferably Tyr, X
at position 94 can be any naturally occurring amino acid,
preferably Pro or Gln, more preferably Pro, X at position 126 can
be any naturally occurring amino acid, preferably Leu, X at
position 137 can be any naturally occurring amino acid, preferably
Lys or Glu, more preferably Glu, X at position 147 can be any
naturally occurring amino acid, preferably Asp or Asn, more
preferably Asp, X can be any naturally occurring amino acid, X at
position 163 can be absent or any naturally occurring amino acid,
preferably Thr, X at position 166 can be absent or any naturally
occurring amino acid, preferably Pro, X at position 168 can be any
naturally occurring amino acid, preferably Gly or Ala, more
preferably Ala.
[0118] In a preferred embodiment the L-type ferritin comprises or
consists of murine full length ferritin according to SEQ ID NO: 11
being preferred. Accordingly, preferred structural variants have at
least 70% amino acid identity, more preferably at least 75% amino
acid identity, more preferably at least 80% amino acid identity,
more preferably at least 85% amino acid identity, more preferably
at least 90% amino acid identity, more preferably at least 95%
amino acid identity and in each case at least 70% of the ability of
a murine L-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 11 to be taken up by CD45.sup.+ leukocytes,
preferably M2 macrophages.
[0119] In a preferred embodiment the L-type ferritin comprises or
consists of human full length ferritin according to SEQ ID NO: 13
being preferred. Accordingly, preferred structural variants have at
least 70% amino acid identity, more preferably at least 75% amino
acid identity, more preferably at least 80% amino acid identity,
more preferably at least 85% amino acid identity, more preferably
at least 90% amino acid identity, more preferably at least 95%
amino acid identity and in each case at least 70% of the ability of
a human L-type ferritin consisting of the amino acid sequence
according to SEQ ID NO: 13 to be taken up by CD45.sup.+ leukocytes,
preferably M2 macrophages.
[0120] In a preferred embodiment the alpha haemoglobin comprises or
consists of a minimal mammalian consensus sequence derived from
aligning full length alpha haemoglobins according to SEQ ID NO: 15
Preferred comprise or consist of the amino acid sequence indicated
in SEQ ID NO: 15 and variants thereof having at least 70% amino
acid identity, more preferably at least 75% amino acid identity,
more preferably at least 80% amino acid identity, more preferably
at least 85% amino acid identity, more preferably at least 90%
amino acid identity, more preferably at least 95% amino acid
identity and in each case at least 70% of the ability of an alpha
haemoglobin consisting of the amino acid sequence according to SEQ
ID NO: 15 to be taken up by CD45.sup.+ leukocytes, preferably M1
macrophages.
[0121] In a preferred embodiment the alpha haemoglobin comprises or
consists of a minimal human amino acid sequence derived of human
alpha haemoglobin according to SEQ ID NO: 17. Accordingly,
preferred structural variants have at least 70% amino acid
identity, more preferably at least 75% amino acid identity, more
preferably at least 80% amino acid identity, more preferably at
least 85% amino acid identity, more preferably at least 90% amino
acid identity, more preferably at least 95% amino acid identity and
in each case at least 70% of the ability of an alpha haemoglobin
consisting of the amino acid sequence according to SEQ ID NO: 17 to
be taken up by CD45.sup.+ leukocytes, preferably M1
macrophages.
[0122] In a preferred embodiment the alpha haemoglobin comprises or
consists of a mammalian consensus sequence derived from aligning
full length alpha haemoglobins according to SEQ ID NO: 16.
Accordingly, preferred structural variants have at least 70% amino
acid identity, more preferably at least 75% amino acid identity,
more preferably at least 80% amino acid identity, more preferably
at least 85% amino acid identity, more preferably at least 90%
amino acid identity, more preferably at least 95% amino acid
identity and in each case at least 70% of the ability of an alpha
haemoglobins consisting of the amino acid sequence according to SEQ
ID NO: 16 to be taken up by CD45.sup.+ leukocytes, preferably M2
macrophages.
[0123] In a preferred embodiment the alpha haemoglobin comprises or
consists of human full length alpha haemoglobin according to SEQ ID
NO: 18. Accordingly, preferred structural variants have at least
70% amino acid identity, more preferably at least 75% amino acid
identity, more preferably at least 80% amino acid identity, more
preferably at least 85% amino acid identity, more preferably at
least 90% amino acid identity, more preferably at least 95% amino
acid identity and in each case at least 70% of the ability of a
human full length alpha haemoglobin consisting of the amino acid
sequence according to SEQ ID NO: 18 to be taken up by CD45.sup.+
leukocytes, preferably M2 macrophages.
[0124] In a preferred embodiment the alpha haemoglobin comprises or
consists of a minimal mammalian consensus sequence derived from
aligning full length beta haemoglobins according to SEQ ID NO: 19
and variants thereof having at least 70% amino acid identity, more
preferably at least 75% amino acid identity, more preferably at
least 80% amino acid identity, more preferably at least 85% amino
acid identity, more preferably at least 90% amino acid identity,
more preferably at least 95% amino acid identity and in each case
at least 70% of the ability of a beta haemoglobin consisting of the
amino acid sequence according to SEQ ID NO: 19 to be taken up by
CD45.sup.+ leukocytes, preferably M1 macrophages.
[0125] In a preferred embodiment the alpha haemoglobin comprises or
consists of a minimal human amino acid sequence derived of human
beta haemoglobin according to SEQ ID NO: 21. Accordingly, preferred
structural variants have at least 70% amino acid identity, more
preferably at least 75% amino acid identity, more preferably at
least 80% amino acid identity, more preferably at least 85% amino
acid identity, more preferably at least 90% amino acid identity,
more preferably at least 95% amino acid identity and in each case
at least 70% of the ability of a beta haemoglobin consisting of the
amino acid sequence according to SEQ ID NO: 21 to be taken up by
CD45.sup.+ leukocytes, preferably M1 macrophages.
[0126] In a preferred embodiment the beta haemoglobin comprises or
consists of a mammalian consensus sequence derived from aligning
full length beta haemoglobins according to SEQ ID NO: 20.
Accordingly, preferred structural variants have at least 70% amino
acid identity, more preferably at least 75% amino acid identity,
more preferably at least 80% amino acid identity, more preferably
at least 85% amino acid identity, more preferably at least 90%
amino acid identity, more preferably at least 95% amino acid
identity and in each case at least 70% of the ability of an beta
haemoglobins consisting of the amino acid sequence according to SEQ
ID NO: 20 to be taken up by CD45.sup.+ leukocytes, preferably M2
macrophages.
[0127] In a preferred embodiment the beta haemoglobin comprises or
consists of human full length beta haemoglobin according to SEQ ID
NO: 22. Accordingly, preferred structural variants have at least
70% amino acid identity, more preferably at least 75% amino acid
identity, more preferably at least 80% amino acid identity, more
preferably at least 85% amino acid identity, more preferably at
least 90% amino acid identity, more preferably at least 95% amino
acid identity and in each case at least 70% of the ability of a
human full length beta haemoglobin consisting of the amino acid
sequence according to SEQ ID NO: 22 to be taken up by CD45.sup.+
leukocytes, preferably M2 macrophages.
[0128] In a preferred embodiment the transferrin comprises or
consists of a mammalian consensus sequence derived from aligning
full length alpha haemoglobins according to SEQ ID NO: 25. Thus,
particularly, preferred transferrins comprise or consist of the
amino acid sequence indicated in SEQ ID NO: 25 and of variants
thereof having at least 70% amino acid identity, more preferably at
least 75% amino acid identity, more preferably at least 80% amino
acid identity, more preferably at least 85% amino acid identity,
more preferably at least 90% amino acid identity, more preferably
at least 95% amino acid identity and in each case at least 70% of
the ability of a transferrin consisting of the amino acid sequence
according to SEQ ID NO: 25 to be taken up by CD45.sup.+ leukocytes
preferably M1 macrophages.
[0129] In a preferred embodiment the transferrin comprises or
consists of human transferrin according to SEQ ID NO: 28.
Accordingly, preferred structural variants have at least 70% amino
acid identity, more preferably at least 75% amino acid identity,
more preferably at least 80% amino acid identity, more preferably
at least 85% amino acid identity, more preferably at least 90%
amino acid identity, more preferably at least 95% amino acid
identity and in each case at least 70% of the ability of a
transferrin consisting of the amino acid sequence according to SEQ
ID NO: 28 to be taken up by CD45.sup.+ leukocytes, preferably M1
macrophages.
[0130] The iron binding properties of transferrins are dependent on
a N-terminally and C-terminally located domain. Thus, in a
preferred embodiment the transferrin used in the present invention
comprises at least the N-terminal domain according to SEQ ID NO: 23
and the C-terminal domain according to SEQ ID NO: 24. Preferred
transferrin comprise proteins that comprise the amino acid sequence
indicated in SEQ ID NO: 23 and 24 as well as variants thereof
having at least 70% amino acid identity, more preferably at least
75% amino acid identity, more preferably at least 80% amino acid
identity, more preferably at least 85% amino acid identity, more
preferably at least 90% amino acid identity, more preferably at
least 95% amino acid identity and in each case at least 70% of the
ability of a transferrin consisting of the amino acid sequence
according to SEQ ID NO: 23 and 24 to be taken up by CD45.sup.+
leukocytes, preferably M1 macrophages. SEQ ID NO: 23 or 24
indicates a consensus sequence of mammalian transferrins.
[0131] Thus, in a preferred embodiment the transferrin used in the
present invention comprises at least the N-terminal domain
according to SEQ ID NO: 26 and the C-terminal domain according to
SEQ ID NO: 27. Preferred transferrin comprise proteins that
comprise the amino acid sequence indicated in SEQ ID NO: 26 and 27
as well as variants thereof having at least 70% amino acid
identity, more preferably at least 75% amino acid identity, more
preferably at least 80% amino acid identity, more preferably at
least 85% amino acid identity, more preferably at least 90% amino
acid identity, more preferably at least 95% amino acid identity and
in each case at least 70% of the ability of a transferrin
consisting of the amino acid sequence according to SEQ ID NO: 26
and 27, respectively, to be taken up by CD45.sup.+ leukocytes,
preferably M1 macrophages.
[0132] Preferred ferritins, also comprise proteins that,
irrespective of the given amino acid sequence, conform to the
24-mer subunit assembly of a four helix bundle protein module,
falling within given sequence alignments of distantly related
proteins as defined by 3D structure based alignments.
[0133] It is a surprising observation of the present inventors that
monocytes, macrophages and preferably M2 macrophages are able to
uptake the amount of label enough to make them visible using
imaging methods at the site of their accumulation (e.g. in the
tumour or its metastasis, hypoxia site). Surprisingly, inventors
found out that lymphocytes and M2 macrophages are better in uptake
of complexes comprising one or more ferritin and one or more
pharmaceutically active substance, label or pharmaceutically active
substance and label, that M1 macrophages are better in uptake of
complexes comprising one or more haemoglobin and one or more
pharmaceutically active substance, label or pharmaceutically active
substance and label and that macrophages are better in uptake of
complexes comprising one or more transferrin and one or more
pharmaceutically active substance, label or pharmaceutically active
substance and label. Accordingly, based on the tissue and cellular
tropism of CD45.sup.+ leukocytes: monocytes, M1 and M2 macrophages,
granulocytes and lymphocytes, described above complexes comprising
one or more ferritin and one or more pharmaceutically active
substance, label or pharmaceutically active substance and label are
used to load M2 macrophages, lymphocytes or monocytes if the
tropism of M2 macrophages, lymphocytes or monocytes is desired and
complexes comprising one or more haemoglobin and one or more
pharmaceutically active substance, label or pharmaceutically active
substance and label are used to load M1 macrophages, if the tropism
of M1 macrophages is desired.
[0134] In a preferred embodiment of the targeted delivery system of
the present invention the pharmaceutically active substance, label
or pharmaceutically active substance and 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 kD, more preferably less than 1 kD, preferably an
anticancer drug, in particular a cytostatic drug, cytotoxic drug
and prodrugs thereof; an anti arteriosclerotic drug; and
anti-inflammatory drug; and photosensitizing compound; a virus, in
particular oncolytic virus; and .alpha. a or radiation emitting
radioisotope, which also emit a cell damaging amount of .gamma.
radiation, preferably 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.
[0135] Preferred anticancer drugs are selected from an
apoptosis/autophagy or necrosis-inducing drug. An
apoptosis/autophagy or necrosis-inducing drug can be any drug that
is able to induce apoptosis/autophagy or necrosis effectively even
in cells having an abnormality in cell proliferation. These drugs
are preferably used in complexes with one or more ferritins.
[0136] Preferred anticancer drugs are 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 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 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.
[0137] Other preferred anticancer drugs are selected from the group
consisting of acediasulfone, aclarubicine, ambazone,
aminoglutethimide, L-asparaginase, azathioprine, banoxantrone,
bendamustine, bleomycin, busulfan, calcium folinate, carboplatin,
carpecitabine, carmustine, celecoxib, chlorambucil, cis-platin,
cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin
dapsone, daunorubicin, dibrompropamidine, diethylstilbestrole,
docetaxel, doxorubicin, enediynes, epirubicin, epothilone B,
epothilone D, estramucin phosphate, estrogen, ethinylestradiole,
etoposide, flavopiridol, floxuridine, fludarabine, fluorouracil,
fluoxymesterone, flutamide fosfestrol, furazolidone, gemcitabine,
gonadotropin releasing hormone analog, hexamethylmelamine,
hydroxycarbamide, hydroxymethylnitrofurantoin,
hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine,
ifosfamide, interferon .alpha., irinotecan, leuprolide, lomustine,
lurtotecan, mafenide sulfate olamide, 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, salazosulfapyridine, scriflavinium
chloride, semustine streptozocine, sulfacarbamide, sulfacetamide,
sulfachlopyridazine, sulfadiazine, sulfadicramide,
sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine,
sulfaguanole, sulfamethizole, sulfamethoxazole, co-trimoxazole,
sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole,
sulfanilamide, sulfaperin, 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, cyclophosphamide
dynemycin A, maytansine, melphalan, mertansine, and
neocazinostatin, most preferably banoxantrone, bendamustine,
chlorambucil, cyclophosphamide, pyrrolobenzodiazepine and
melphalan.
[0138] It is particularly preferred that 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 DNAzym.
[0139] Preferred immunomodulatory drugs that activate or inhibit
activity of immune cells. These can be natural or synthetic ligands
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).
[0140] 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 (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.
[0141] It has been surprisingly found by the present inventors that
inflamed joints are targeted by macrophage-monocytes, preferably
activated macrophages. Thus, in diagnostic applications in which
inflamed joints are to be detected macrophage-monocytes are
preferably loaded with a label or a complex comprising an iron
binding protein and a label. In therapeutic applications, it is
preferred that the complex comprises a pharmaceutically active
substance with anti-inflammatory activity to deliver the
anti-inflammatory substance to the inflamed tissue.
[0142] It has been surprisingly found by the present inventors that
the targeted delivery system of the present invention targets lymph
nodes, which makes it particularly suitable 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
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 group consisting of
epidermal growth factor receptor (EGFR, ErbB-1, HER1), 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) 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 .alpha. (TGF-.alpha.) receptors, TGF-.beta.; Cytokine
receptors, Class I (hematopoietin family) and Class II
(interferon/IL-10 family) receptors, tumor necrosis factor (TNF)
receptor superfamily (TNFRSF), death receptor family; cancertestis
(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),
.beta.-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 gene
6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein,
fibronectin (FN), GPNMB, low density lipid receptor/GDP-L fucose:
.beta.-Dgalactose 2-.alpha.-Lfucosyltransferase (LDLR/FUT) fusion
protein, HLA-A2. arginine to isoleucine exchange at residue 170 of
the .alpha.-helix of the .alpha.2-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),
MAGE-A1 (MAGE-1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1,
MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1),
MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (SILV), tyrosinase
(TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17,
SSX-1,2,3,4, TRP2-INT2, carcino-embryonic antigen (CEA), Kallikrein
4, mammaglobin-A, OA1, 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 (EpCAM), EphA3, fibroblast growth factor-5 (FGF-5),
glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu (ERBB2),
interleukin 13 receptor .alpha.2 chain (IL13Ralpha2), IL-6
receptor, intestinal carboxyl esterase (iCE), alpha-feto protein
(AFP), M-CSF, 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, CSAGE,
MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC, MORC, SGY-1,
SPO11, 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), .beta.-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) proteins (EBV latent membrane
proteins-LMPL LMP2; others), Hepatitis B or C virus proteins, and
HIV proteins.
[0143] If the pharmaceutically active substance is a nucleic acid
it is preferred that it is a miRNA, siRNA, chemically modified-RNA,
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.
[0144] In a preferred embodiment of the targeted delivery system of
the present invention the label is selected from a fluorescent dye,
a fluorescence emitting isotope, a radioisotope, a detectable
polypeptide or nucleic acid encoding such a detectable polypeptide
and a contrast agent.
[0145] In a preferred embodiment of the targeted delivery system of
the present invention the label comprises a chelating agent which
forms a complex with divalent or trivalent metal cations.
[0146] 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,
Diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA) and
6-Hydrazinopyridine-3-carboxylic acid (HYNIC).
[0147] In a preferred embodiment of the targeted delivery system of
the present invention the contrast agent comprises a paramagnetic
agent, preferably selected from Gd, Eu, W and Mn, or
ferrihydride.
[0148] In a preferred embodiment of the targeted delivery system of
the present invention 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, fluorescence
emitting isotopes, such as .sup.18F, .sup.51Cr, fluorescent
.sup.65Tb, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.99mTc,
.sup.140La, .sup.175Yb, .sup.153Sm, .sup.166Ho, .sup.88Y, .sup.90Y,
.sup.149Pm, .sup.177Lu, .sup.47Sc, .sup.142Pr, .sup.159Gd,
.sup.212Bi, .sup.72As, .sup.72Se, .sup.97Ru, .sup.109Pd,
.sup.105Rh, .sup.101m15Rh, .sup.119Sb, .sup.128Ba, .sup.123I,
.sup.124I, .sup.131I, .sup.197Hg, .sup.211At, .sup.169Eu,
.sup.203Pb, .sup.212Pb, .sup.64Cu, .sup.89Zr, .sup.67Cu,
.sup.188Re, .sup.186Re, .sup.198Au and .sup.199Ag as well as
conjugates and combinations of above with proteins, peptides, small
molecular inhibitors, antibodies or other compounds (e.g.
.sup.18F-FDG, .sup.89Zr-oxide or .sup.64Cu-porfirin).
[0149] In a preferred embodiment of the targeted delivery system of
the present invention the fluorescence 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.
[0150] In a preferred embodiment of the targeted delivery system of
the present invention the detectable polypeptide is an
autofluorescent protein, preferably green fluorescent protein or
any structural variant thereof with an altered adsorption and/or
emission spectrum.
[0151] As has been outlined above, the targeted delivery system of
the present invention has particular suitability to deliver
pharmaceutically active substances, labels or pharmaceutically
active substances and labels to hypoxic areas. Hypoxy is
characteristic of various disease including cancer and inflammatory
diseases and thus allows targeting such diseases.
[0152] In addition to the targeting the use of pharmaceutically
active substances, which are activated under hypoxic conditions
adds a further specificity to the targeting and/or further reduces
adverse effects of the pharmaceutically active substances, labels
or pharmaceutically active substances and labels. Thus, in
particularly preferred embodiments the pharmaceutically active
substance, label or pharmaceutically active substance and label 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
prodrugs 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 PR104A,
TH-302, TH-4000, and AQ4N.
[0153] In a preferred embodiment of the isolated targeted delivery
system of the present invention the bond(s) between the iron
binding protein(s) and the pharmaceutically active substance, label
or pharmaceutically active substance and label comprised in the
complex are covalent and/or non-covalent; and/or the
pharmaceutically active substance, label or pharmaceutically active
substance and label comprised in the complex is
entrapped/encapsulated by the iron binding protein, preferably
ferritin or multimers thereof. 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 iron binding proteins are
used to covalently couple pharmaceutically active substances,
labels or pharmaceutically active substances and labels directly or
indirectly to the one or more iron binding protein.
[0154] It is also envisioned that different pharmaceutically active
substances, labels or pharmaceutically active substances and labels
are comprised in the complex. For example, one type of
pharmaceutically active substance, label or pharmaceutically active
substance and label may be bound to an iron binding protein
(non-covalently bound), while another type is encapsulated. This
approach utilizes different release rates of the pharmaceutically
active substances, labels or pharmaceutically active substances and
labels from the iron binding protein once delivered to the targeted
tissue and/or cells. For example, pharmaceutically active
substance, label or pharmaceutically active substance and label can
be covalently attached to 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
pharmaceutically active substance, label or pharmaceutically active
substance and label without any additional work. Examples of such
reactions are described in Behrens C R, Liu B. Methods for
site-specific drug conjugation to antibodies. MAbs. 2014 Jan-Feb;
6(1):46-53.
[0155] In theragnostic applications, i.e. in which the complex
comprises both a label and a pharmaceutically active substance, it
is preferred that the label is covalently attached to the iron
binding protein and the pharmaceutically active substance is
non-covalently bound to the iron binding protein and/or entrapped
in the internal cavity formed upon assembly of the iron binding
proteins.
[0156] In a third aspect the present invention relates to a method
of preparation of the isolated targeted delivery system of the
first or second aspect comprising the steps of [0157] a) providing
purified iron binding protein; [0158] b) covalently or
non-covalently linking a pharmaceutically active substance, label
or pharmaceutically active substance and label to and/or
encapsulating a pharmaceutically active substance, label or
pharmaceutically active substance and label in an iron binding
protein; [0159] c) providing a CD45.sup.+ leukocyte cell; and
[0160] d1) incubating the CD45.sup.+ leukocyte cell in the presence
of the iron binding protein produced in step b) until the
CD45.sup.+ leukocyte cell is at least partially loaded with the
complex of the iron binding protein and the a pharmaceutically
active substance, label or pharmaceutically active substance and
label produced in step b); and/or [0161] d2) incubating CD45.sup.+
leukocyte cell in the presence of the label until the CD45.sup.+
leukocyte cell is at least partially labelled with the label.
[0162] In a further aspect the present invention relates to the
isolated targeted delivery system producible by the method
according to the third aspect of the present invention.
[0163] The formation of the adduct between the protein and the
pharmaceutically active substance and/or label is preferably by a
non-covalent binding to the iron binding protein and can be
described as follows: In the case of ferritin, pharmaceutically
active substances and/or labels can be typically encapsulated
within the internal cavity (physical confinement) by exploiting the
association dissociation properties of the ferritin macromolecule
itself. pharmaceutically active substance and/or label molecules
are held in place by non-covalent interactions with amino acid
residues within the cavity internal surface. Haemoglobin
macromolecules also offer the possibility of non-covalent binding
of selected pharmaceutically active substances and/or labels
molecules that may be hosted within the heme binding pocket of
haemoglobin itself. The heme in the pocket can be displaced and be
replaced by pharmaceutically active substances and/or labels with
appropriate hydrophobicity profile. In a further aspect, all
proteins considered in the present invention may be covalently
attached to pharmaceutically active substance and/or label
molecules modified by specific active linker moieties reactive
towards thiol or amino groups of the protein itself. As such,
ferritins or haemoglobin may be linked to cysteine thiol reactive
pharmaceutically active substances and/or labels bearing a peptide
based cleavable linker (e.g. cathepsin sensitive valine-citrulline
sequence and para-aminobenzylcarbamate spacer). As a notable
example, the antimitotic agent monomethyl auristatin 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
protein pharmaceutically active substance and/or label adduct thus
generated is capable of attaching to the selected receptor types,
i.e. CD163 for haemoglobin and TfR for ferritin or transferrin,
respectively. Once bound the protein pharmaceutically active
substance and/or label adduct is internalised by endocytosis and
thus 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 valine-citrulline
linker and MMAE is no longer bound to the iron binding protein and
is released directly into the tumour environment.
[0164] Alternatively, DM1-SMCC is an efficient mertansine
derivative bearing a linker that specifically binds to lysine
residues generating a covalent complex with ferritin, haemoglobin
or transferrin in a reaction that has been successfully described
for antibodies. In particular, haemoglobin, ferritin or transferrin
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.
[0165] The term "full load" is used in the context of the present
invention to refer to the maximum amount of iron binding protein,
preferably ferritin, complexed with a pharmaceutically active
substance, label or pharmaceutically active substance and label
that can be taken up by the CD45+ leukocyte cell, preferably
macrophage more preferably activated macrophage.
[0166] In a fourth aspect the present invention relates to an
isolated targeted delivery system of the first aspect or second
aspect of the invention or producible according to the method of
the third aspect of the invention for use as a medicament or
diagnostic.
[0167] In a fifth aspect the present invention relates to a
pharmaceutical or diagnostic composition comprising the isolated
targeted delivery system of the first or second aspect of the
present invention or producible according to the method of the
third aspect of the invention and a pharmaceutically acceptable
carrier and/or suitable excipient(s). Since the isolated targeted
delivery system comprises living cells, it is preferred that
carriers and excipients are chosen in such to keep the cells
alive.
[0168] "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.
[0169] 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.
[0170] 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.
[0171] "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.
[0172] "Stabilizers" include but are not limited to mannitol,
sucrose, trehalose, albumin, as well as protease and/or nuclease
antagonists.
[0173] "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), HEPES 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.
[0174] 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 (e.g. saponins or squalene),
oil-based adjuvants (e.g. Freund's complete adjuvant and Freund's
incomplete adjuvant), cytokines (e.g. IL-1.beta., IL-2, IL-7,
IL-12, IL-18, GM-CFS, and INF-.gamma.) 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).
[0175] A striking observation was the disease specific homing of
the targeted delivery system of the present invention. The
CD45.sup.+ 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, in a sixth
aspect the present invention relates to an isolated targeted
delivery system of the first aspect or second aspect of the present
invention or producible according to the method of the third aspect
of the invention for use in preventing, treating or diagnosing a
disease characterized by hypoxic areas within the diseases tissue
and/or by areas of oxidative stress (FIG. 27); tumours, preferably
solid tumours and/or its metastases, preferably breast cancer,
pancreatic cancer, bladder cancer, lung cancer, colon cancer, or a
tumour having hypoxic areas, an inflammatory disease, inflamed
tissue, preferably inflamed joints or arthritic joints, inflamed
lung, inflamed intestine or other inflamed tissue; lymph nodes,
preferably inflamed lymph nodes, or other non-physiological lymph
nodes that develop during disease preferably but not only during
infection, cancer, or autoimmune 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 also of antigens to physiological or
non-physiological lymph nodes in order to vaccinate an individual
or to induce immune memory.
[0176] 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.
[0177] 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). It also enables the delivery
of pharmaceutically active substances, labels or pharmaceutically
active substances and labels to the hypoxic tumours or to the
hypoxic areas of the tumour. This system also provides for delivery
of pharmaceutically active substance, label or pharmaceutically
active substance and label to any area within an organism subjected
to hypoxic conditions, for example during ischaemic incidents, or
undergoing an inflammatory process.
[0178] As mentioned above the present invention provides also the
method for targeted delivery into the tumour mass. This method
comprises preparation of CD45.sup.+ leukocytes, preferably
activated macrophages which enables highly efficient iron-binding
protein (ferritin, haemoglobin and/or transferring) uptake by the
macrophages, wherein said ferritin, haemoglobin and/or transferrin
carry a pharmaceutically active substance, label or
pharmaceutically active substance and label (for example a
drug/prodrug), tumour targeting and iron-binding protein transfer
to the cancer cell, where the pharmaceutically active substance,
label or pharmaceutically active substance and label is
released.
[0179] The present invention exploits CD45.sup.+ leukocytes,
preferably activated macrophages loaded with iron-binding proteins
linked with a drug/prodrug as a delivery system to target the
tumour. Unsatisfactory response of the tumours to chemotherapy or
difficulties in their detection using imaging methods are mainly
related to an altered penetration of the anticancer drugs to the
hypoxic areas due to poor vasculature. However, these avascular
regions attract CD45.sup.+ leukocytes, preferably activated
macrophages to migrate even in areas far away from blood vessels.
Therefore, they constitute a delivery system of particles to the
tumour mass. A promising example of such particles is iron-binding
protein. However, when used as single agents they do not reach
hypoxic regions, similarly to other compounds and accumulate in
other organs.
[0180] The present inventors linked anticancer drugs, hypoxia
activated prodrugs (for treatment purposes), labels or isotopes to
haemoglobin or transferrin using chemical methods and loaded it
into CD45.sup.+ leukocytes (monocytes, macrophages, lymphocytes
and/or granulocytes), preferably activated macrophages treating
cells with iron-binding protein solution as it is described in
examples. The inventors observed that upon administration to the
animal, loaded CD45.sup.+ leukocytes, preferably activated
macrophages migrate to the tumour hypoxic sites and release
iron-binding protein with encapsulated pharmaceutically active
substances, labels or pharmaceutically active substances and labels
into the cancer cells. This method allows precise administration of
the pharmaceutically active substances, labels or pharmaceutically
active substances and labels to the tumour site (especially to the
hypoxic regions), avoiding their accumulation in other organs.
BRIEF DESCRIPTION OF DRAWINGS
[0181] FIG. 1: Panel (A) shows a minimal active fragment of a
consensus amino acid sequence among mammalian ferritin H chains and
two full length consensus sequences based on several mammalian
ferritin H chains (see SEQ ID NO: 1, 2 and 7, respectively) as well
as a minimal and full length amino acid sequence of mouse (SEQ ID
NO: 3 and 4) and human (SEQ ID NO: 5 and 6) ferritin H chain. Panel
(B) shows a minimal active fragment of a consensus amino acid
sequence among mammalian ferritin L chains and two full length
consensus sequences based on several mammalian ferritin L chains
(see SEQ ID NO: 8, 9 and 14, respectively) as well as a minimal and
full length amino acid sequence of mouse (SEQ ID NO: 10 and 11) and
human (SEQ ID NO: 12 and 13) ferritin L chain. Panel (C) shows a
minimal active fragment of a consensus amino acid sequence among
mammalian haemoglobin alpha chains and one full length consensus
sequences based on several mammalian haemoglobin alpha chain (see
SEQ ID NO: 15 and 16, respectively) as well as a minimal and full
length amino acid sequence of human (SEQ ID NO: 17 and 18)
haemoglobin alpha chain. Panel (D) shows a minimal active fragment
of a consensus amino acid sequence among mammalian haemoglobin beta
chains and a full length consensus sequences based on several
mammalian haemoglobin beta chain (see SEQ ID NO: 19 and 20,
respectively) as well as a minimal and full length amino acid
sequence of human (SEQ ID NO: 21 and 22) haemoglobin beta chain.
Panel (E) shows a N- and C-terminal minimal active fragment of a
consensus amino acid sequence among mammalian transferrins (SEQ ID
NO: 23 and 24) and a full length consensus sequences based on
several mammalian transferrins (SEQ ID NO: 25) as well as a N- and
C-terminal minimal active fragment of a human transferrin (SEQ ID
NO: 26 and 27) and full length amino acid sequence of human
transferrin (SEQ ID NO: 28). In the consensus sequences X indicates
a position that is variable and stands for any natural amino acid.
Preferably, in each case X in dependently of other X stands for the
amino acid present in the human protein.
[0182] FIG. 2: Shows macrophage inside the mouse tumour mass (TRITC
stained before injection, loaded with FITC-decorated ferritin).
[0183] FIG. 3: Shows confocal microscopy image of tumour tissue
mouse injected with mammary cancer cells and given i.v. macrophages
loaded with FITC-ferritin (asterix)--it is clearly observed, not
only in macrophages but also in cancer cells, that ferritin-FITC
spread within all tumour mass.
[0184] FIG. 4: Shows snapshots of one channel (in original green
channel converted to the grey-scale picture) recording using
confocal microscopy of macrophages (indicated with *; loaded with
FITC-ferritin) and cancer cell (indicated with arrow; stained with
red label and therefore not observed in green channel before
ferritin uptake) in vitro taken at the starting time point (A) and
after time long enough to fill cancer cell with ferritin (B).
FITC-ferritin was dynamically transported to the cancer cell
(accumulating firstly in the vesicles; then spreading to the whole
cytoplasm as seen at this image (the cell appeared in green
channel).
[0185] FIG. 5: Shows survival of mice receiving placebo and
macrophages loaded with ferritin-coupled melphalan and
ferritin-coupled chlorambucil.
[0186] FIG. 6: Shows tumour cells apoptosis caused by treatment
with cyclophosphamide and cyclophosphamide encapsulated in
ferritins loaded to macrophages (given at the same doses).
[0187] FIG. 7: Shows MRI images of mouse mammary tumour. The mouse
was treated (at time point 0 h) with macrophages (i.v. injection)
loaded with ferritin Fh. Then we observed increased diameter of
blood vessels (arrow) filled with injected macrophages (giving
significant T2-signal reduction) and afterword macrophages spread
to the tissue (spot-like pattern; arrows). These changes (in the
same time points) were observed in all examined mice.
[0188] FIG. 8: Shows ferritin, haemoglobin and transferrin uptake
by macrophages, ferritin and haemoglobin uptake by monocytes and
ferritin uptake by lymphocytes and granulocytes.
[0189] FIG. 9: Shows the stability of the ferritin storage by
macrophages.
[0190] FIG. 10: Shows transfer of ferritin, haemoglobin and
transferrin from macrophage to various cancer cells.
[0191] FIG. 11: Shows the transfer of ferritin from macrophage to
cancer and non-cancer cells.
[0192] FIG. 12: Shows the transfer of ferritin encapsulated with
hypoxia activated prodrug from macrophage to cancer cells.
[0193] FIG. 13: Shows the apoptosis in cancer cells that received
ferritin with encapsulated various anticancer agents from
co-cultured macrophages or soluble ferritin with the same
agents.
[0194] FIG. 14: Shows the transfer of ferritin, haemoglobin and
transferrin from monocyte to various cancer cells.
[0195] FIG. 15: Shows the transfer of ferritin and haemoglobin from
granulocyte to various cancer cells.
[0196] FIG. 16: Shows the transfer of ferritin and haemoglobin from
lymphocyte to various cancer cells.
[0197] FIG. 17: Shows the picture from two-photon microscopy
showing tumour from a mouse that received pre-labeled (before
administration) macrophages containing Ferritin-FITC.
[0198] FIG. 18: Shows the whole body imaging of mice that
intravenously received labeled macrophages, showing their
accumulation in the tumour site and their distribution in other
organs.
[0199] FIG. 19: Shows the migration of macrophages to hypoxic
tissue, a cross-section of the tumour from a mouse that was
administered intravenously with pre-labeled macrophages, tumour
hypoxic areas are visualized with a hypoxia
marker--pimonidazolone.
[0200] FIG. 20: Shows the presents localization of vesicles
containing FITC-laded ferritin (round objects) in the
microenvironment inside the tumour mass. Macrophages containing
FITC-laded ferritin were administered intravenously to the
mouse.
[0201] FIG. 21: Shows the signal recorded by PET from a whole-body
analysis of mice with the metastatic 4T1 cancer. Mice received
intravenously macrophages loaded with .sup.18F-FDG. Signal
accumulation is increased in the lungs of mice with micrometastases
(confirmed by pathology examination). Mice receiving plain
.sup.18F-FDG, or mice without 4T1 cancer had lower PET signal.
MQ-FDG: indicates mice with metastatic 4T1 tumour+intravenous
macrophages loaded with .sup.18F-FDG. FDG: indicates mice with
metastatic 4T1 tumour+intravenous free .sup.18F-FDG. Naive mice
MQ-FDG: Indicates naive mice without tumour+intravenous macrophages
loaded with .sup.18F-FDG.
[0202] FIG. 22: Shows the tumour homing by different CD45+
leukocyte cell types usable in the targeted delivers systems of the
invention. Mice with CT26 tumors (marked by the arrows) were
intravenously injected with PBS or fluorescently labelled cells.
Panel A. --PBS, no cells added, Panel B.--shows homing of a
CD45.sup.+ leukocyte subpopulation, Panel C. shows homing of
activated CD4.sup.+ T lymphocytes, Panel D. shows homing of a
macrophage-monocytic cell line. 24 hours after injection mice were
anaesthetized and imaged. Panel E. Shows the quantification of the
tumour homing by different CD45+ leukocyte cells.
[0203] FIG. 23: Shows Survival of mice with CT26 tumor receiving
therapy with monocytes loaded with ferritin-Cisplatin. n=6-8
mice/group. Survival of the mice was calculated by reaching (1500
mm3) maximum-allowed tumour volume (measured by caliper, according
to formula A.times.B2).
[0204] FIG. 24: Shows targeting of intravenously administered cells
to inflamed (arthritic) joints Control mouse (panel A.) or a mouse
with severe inflammation of the joints (panel B.) have been
intravenously injected with macrophage-monocyte cell line. Before
injection cells were labelled with fluorescent tracer. 24 hours
after injection joints were removed and single cell suspension
generated. Cells have been analyzed by flow cytometry.
Fluorescently labelled cells are presented in the P2 area. Panel C.
shows quantification of the fluorescent cells in the inflamed than
in control joints. Macrophage-monocyte cells are present with
greater number in the inflamed than in control joints.
[0205] FIG. 25: Shows targeting of intravenously administered cells
to the lymph nodes. Control mouse (panel A.) or a mouse with severe
inflammation of the joints (panel B.) have been intravenously
injected with activated macrophages (with M-CSF and CCL2). Before
injection cells were labelled with fluorescent tracer. 24 hours
after injection mice were anaesthetized and imaged. Mice with
inflammatory condition have positive signal derived from
cell-tracer, from the lymph nodes (marked by white arrows).
[0206] FIG. 26: Shows radiolabeling of monocyte-macrophage cell
line with plain .sup.89Zr-oxinate, i.e. without iron binding
protein (.about.1.85 MBq) for PET imaging. Radioactivity of cells
after loading with .sup.89Zr is indicated in MBq.
[0207] FIG. 27: Ferritin transfer was examined in cancer cells
(EMT6) co-cultured with ferritin-loaded macrophages (RAW264.7) for
2 hrs. Three experimental conditions were used: "ctrl" (normal
co-culture, no stress); "EMT6 OS" when EMT6 cells were subjected to
the oxidative stress before co-culture and then co-cultured with
macrophages at normoxic conditions and "co-culture OS" when both
cell types were subjected to the oxidative stress before the
co-culture. Results showed that cancer cells subjected to the
oxidative stress received (from macrophages) more ferritins than
control cells. The uptake was even more efficient when both cell
types were subjected to the oxidative stress. It can be related
with higher load of ferritin due to oxidative stress and therefore
more efficient transfer, or oxidative stress triggers macrophages
to more efficiently deliver ferritins to cancer cells in order to
reduce oxidative stress in neighboring cells. Panel A shows:
Ferritin uptake from macrophages (2 hrs co-culture) in oxidative
stress (oxidative glucose treatment for 16 hrs). Panel B shows:
Ferritin uptake from macrophages (2 hrs co-culture) in oxidative
stress (oxidative glucose treatment for 16 hrs).
EXAMPLE SECTION
Example 1--Activation of Macrophages
[0208] Macrophages for use according to the present invention were
obtained, differentiated and activated as follows. In order to
activate macrophages, they are obtained firstly from bone marrow
precursors (for example see paper: Weischenfeld and Porse, 2008,
CSH Protoc, doi. 10.1101/pdb.prot.5080) or blood monocytes.
Alternatively, they can be obtained from peritoneum. The methods of
macrophage isolation, culture, differentiation and
polarization/activation are well known for those skilled in the
art. For example, they have been described in details by Murray et
al. (Immunity, 2014, 41(1):14-20).
[0209] In this practical realization of the invention bone marrow
derived macrophages were obtained from BALB/c or C57Bl/6 mouse,
however canine blood-monocyte-derived macrophages or commercially
available macrophage cell lines (monocyte-macrophage lineage mouse
cells: RAW 264.7, J744, human: THP-1, U937, or canine DH82 cell
line).
[0210] Shortly, such bone marrow derived macrophages are seeded in
plastic Petri dish in 5 ml medium (3 ml cells per plate):
DMEM:F12+glutamine/glutamax+10% FBS+Penicillin/Streptomycin and 20%
of L929 conditioned medium or M-CSF (50 ng/ml). In the next five
days the medium is supplemented in growth factor and one of the
activating compounds or their combinations as one cytokine
cocktail.
[0211] Alternatively, macrophages have been cultured in "M1/M2
Macrophage Generation Medium" (Promocell) or equivalent
commercially available or self-made medium containing all the
necessary cytokines and interleukins to consider them as
activated.
[0212] In order to obtain macrophages from blood monocytes, fresh
blood (not older than 12 hours) cells were purified by
centrifugation using Histopaque system 1.077 g/ml or equivalent and
white blood cells (or alternatively, only white blood cells
collected from the blood bank) in an appropriate amount of
pre-warmed Monocyte Attachment Medium (or equivalent, e.g.
DMEM/RPMI supplemented with M-CSF), e.g. 15 ml Medium per T-75
flask. A seeding density should be of 1-2 million/cm.sup.2 for
mononuclear cells with a monocyte content of .gtoreq.2 5% and 1.5-3
million/cm.sup.2 for a monocyte content of <25%. Then, cells are
incubated for 1-1.5 hours at 5% CO.sub.2 and 37.degree. C. in the
incubator without any further manipulation.
[0213] After cell attachment, they are washed at least twice, and
then an appropriate amount of complete "M1- or M2-Macrophage
Generation Medium DXF" is added to the cells (e.g. 20 ml per T-75
flask) and cells are incubated for 6 days at 37.degree. C. and 5%
CO.sub.2 without medium change. In order to activate macrophages,
the whole medium is replaced with medium supplemented with
activating compound.
[0214] Activating compounds used in this invention (for bone-marrow
derived cells or to activate cells from monocyte-macrophage cell
lines) are as follows: IL-4 (20 ng/ml), IFN-.gamma. (at least 20
ng/ml), LPS (at least 10 ng/ml), IL-13 (at least 20 ng/ml), IL-10
(at least 20 ng/ml), dexamethason (at least 20 .mu.g/ml), CCL2 (at
least 20 ng/ml), oxLDL (at least 20 ng/ml), TNF-.alpha. (20 ng/ml),
TGF-.beta. (20 ng/ml), cortisol (150-300 ng/ml) or their
combinations as one cytokine cocktail. In order to obtain
non-activated macrophages, the activating compound has not been
added.
[0215] Reverse of the polarization/activation of macrophages (from
classically activated to alternatively activated) can be reached
for example by culture of macrophages in appropriate cytokines
listed above for at least 48 hours.
Example 2--Monocyte Isolation
[0216] In order to obtain monocytes in this practical realization
of the invention bone marrow derived or spleen-derived monocytes
were obtained from BALB/c or C57Bl/6 mouse, however canine blood
monocyte or commercially available monocyte cell lines were used
(monocyte-macrophage lineage mouse cells: RAW 264.7, J744, human
cells: THP-1, U937, or canine cells DH82).
[0217] To obtain blood monocytes, fresh blood (not older than 12
hours) cells were purified by centrifugation using Histopaque
system 1.077 g/ml or equivalent and white blood cells are seeded in
an appropriate amount of pre-warmed Monocyte Attachment Medium (or
equivalent, e.g. DMEM/RPMI supplemented with 20 ng/ml M-CSF), e.g.
15 ml Medium per T-75 flask. Alternatively, only white blood cells
collected from the blood bank (buffy coat) may be used. After cell
attachment, they are washed at least twice, and adherent cells are
considered as monocytes.
[0218] In order to obtain bone-marrow derived monocytes, in this
practical realization of the invention monocytes were obtained from
BALB/c or C57Bl/6 mouse. Shortly, such bone marrow derived
precursors were isolated using Mouse Monocyte Enrichment Kit
(StemCells) according to manufacturer's instructions. In order to
obtain spleen derived monocytes, in this practical realization of
the invention, the spleen has been mechanically dissociated to
obtain single cell suspension and passed through the 70 .mu.m cell
strainer. Cells were centrifuged and supernatant was removed.
Optionally erythrocyte lysis was performed prior to monocyte
isolation by EasySep.TM. Mouse Monocyte Enrichment Kit (StemCells)
according to manufacturer's instructions.
[0219] To obtain better effects of their protein loads and
migration before use they may be pre-treated with activation
stimuli: IL-4 (20 ng/ml), IFN-.gamma. (at least 20 ng/ml), LPS (at
least 10 ng/ml), IL-13 (at least 20 ng/ml), IL-10 (at least 20
ng/ml), dexamethason (at least 20 .mu.g/ml), CCL-2 (at least 20
ng/ml), oxLDL (at least 20 ng/ml), TNF-.alpha. (20 ng/ml),
TGF-.beta. (20 ng/ml), cortisol (150-300 ng/ml) or their
combinations as one cytokine cocktail.
Example 3--Granulocyte Isolation
[0220] To obtain granulocyte cells from blood, 9 parts of blood
were diluted with 1 part of ACD buffer (containing 0.17 M
d-glucose, 0.10 M citric acid, 0.11 M trisodium citrate). Blood
from this step was further diluted with PBS at the 1:1 ratio and
centrifuged. After removing plasma and buffy coat, remaining cells
were mixed with PBS to 80% of the original volume from the first
step (ACD-blood) and then diluted with cold distillated water at
the ratio of 4:12. Then, 6 parts of 2.7% of NaCl solution were
added and centrifuged. After removal of supernatant cells were
resuspended in RPMI-1640 medium. These cell were considered as
granulocytes.
Example 4--Lymphocyte Isolation
[0221] In order to obtain spleen derived lymphocytes, in this
practical realization of the invention, the spleen has been
mechanically dissociated to obtain single cell suspension and
passed through the 70 .mu.m cell strainer. Cells were centrifuged
and supernatant was removed. After erythrocyte lysis, lymphocytes
were isolated using a negative cell isolation strategy with a help
of EasySep.TM. CD4.sup.+ Enrichment Kit (Stemcell) according to
manufacturer s instructions.
[0222] Lymphocytes were activated by the 3-5 day culture in RPMI
medium supplemented with glutamine/glutamax, 10% FBS,
Penicillin/Streptomycin, in the presence of beads covered with
anti-CD3 and anti-CD28 (Gibco) according to manufacturer's
instructions.
[0223] Activation of lymphocytes was confirmed with upregulation of
CD25 and CD69 cell surface expression, that was monitored by flow
cytometry.
Example 5--Leukocyte (CD45.sup.+ Cells) Isolation
[0224] In order to obtain macrophages from blood monocytes, fresh
blood (not older than 12 hours) is purified by centrifugation using
Histopaque system 1.077 g/ml or equivalent and white blood cells
(or alternatively, only white blood cells collected from the blood
bank) are used for further steps of the procedure. Alternatively,
leukocytes are obtained after erythrocyte lysis of the whole blood,
as it was done in this practical realization of the invention.
Example 6--Preparation of Ferritin Complexes
[0225] In order to incorporate ferritins with the anticancer drug
(e.g. classic drugs like cyclophosphamide, chlorambucil, melphalan,
bendamustine, banoxantrone or hypoxia-activated prodrug like
TH-302) or with the "imaging contrast agents" (e.g. ferrihydride or
isotope) ferritins have to be prepared before macrophage treatment.
Shortly, recombinant mouse proteins according to SEQ ID NO: 4 (FIG.
1) are obtained as follows. The expression vector pET-22b
containing a synthetic gene encoding ferritin protein of SEQ ID NO:
4 was transformed into E. coli BL21 (DE3). E. coli culture was
grown at 37.degree. C. to OD600 0.6 in 1 L of Luria-Bertani broth
(LB) added with ampicillin (100 mg/L). Protein expression was
induced by addition of 1 mM isopropyl thio-b-D-galactoside (IPTG)
and the culture was incubated overnight. Cells were harvested by
centrifugation (15000 g for 15 min) and suspended in 20 mM Hepes
(pH 7.5), 150 mM NaCl, 0.1 mg/mL DNase, 10 mM MgCl.sub.2 and
disrupted by sonication. The lysate was centrifuged at 15000 g for
30 min and the supernatant was treated 10 min at 50.degree. C.,
centrifuged to remove denatured proteins and then at 70.degree. C.
for 10 min and centrifuged again. The supernatant was added with
30% (NH).sub.4SO.sub.4 at 4.degree. C. stirring for 1 h and
centrifuged at 15000 g for 30 min. The supernatant was added with
70% (NH).sub.4SO.sub.4 at 4.degree. C. stirring for 1 h and
centrifuged at 15000 g for 30 min. The pellet was resuspended in 20
mM Hepes (pH 7.5), 150 mM NaCl and dialysed overnight at 4.degree.
C. against the same buffer. The protein was loaded on a HILOAD
26/600 SUPERDEX 200 gel-filtration column (GE-Healthcare) and then
sterile filtered and stored at 4.degree. C. (FIG. 9) Protein
concentration was determined spectrophotometrically at 280 nm using
a molar extinction coefficient of 21000 M.sup.-1 m.sup.-1 and by
Bradford assay measuring the absorbance at 595 nm.
[0226] Said ferritins include recombinant mammalian ferritin
proteins H and/or L homopolymers.
[0227] Ferritins, obtained as previously described, are purified by
standard methods in order to obtain an endotoxin free, pre-clinical
grade product (see, for example: Ceci et al. 2011, Extremophiles
15(3):431-439; Vanucci et al. 2012, Int J Nanomed 7:1489-1509).
Shortly, the ferritin conserved sterile in a storage solution
containing 20 mM Hepes pH 7.5 is diluted to a final concentration
of 4 uM in 24-mer in acidic solution (final pH<3.0) or,
alternatively, at highly basic pH values (pH>9.5) (see for
example Pontillo et al., 2016), thus allowing the dissociation of
multimer. Drugs are dissolved at very high concentrations in the
appropriate solvent and then a small volume is added to the
ferritin solution with a 200 molar excess. PH is then brought to
neutrality by addition of appropriate amounts of NaOH/HCl solutions
in order to allow multimer reconstitution. Current experimental
methods indicate that three/four washings using PBS (concentration
steps) in 100 kDa cut off concentrators allows rapid and complete
elimination both the co-solvents as well as nonencapsulated drugs
and full recovery of drug loaded ferritin nanocages. The
ferritin-drug complex thus obtained was then flash freezed in
liquid nitrogen and lyophilised.
[0228] Depending on the choice of co-solvent and on the intrinsic
chemical properties of the drug molecule, it can be estimated that
up to 150-180 drug molecules can be entrapped/adsorbed within the
24-mer ferritin cage.
[0229] Pharmaceutically active substances or labels may also be
covalently coupled to ferritin amino acid side chains (lysines or
cysteines) by appropriate choice of phenylhydrazone, succinimide or
maleimide activated drugs. Accordingly, i) phenylhydrazone
derivative may breaks and liberates the drug from the ferritin
surface, ii) lysine bound derivatives may become active after full
protein degradation into aminoacids or iii) cysteine bound
derivative may be liberated within the cell through reductive
hydrolysis of the maleimede thioether link.
Example 7--Preparation of Haemoglobin-Compound Complex
[0230] Human haemoglobin is prepared from fresh red cells as
described in Rossi-Fanelli et al. (Archives of biochemistry and
biophysics 77:478-492, 1958). Shortly, the heparinized blood,
obtained from healthy donors, was centrifuged at 1600 rpm for 30
minutes (4.degree. C.) to sediment the RBCs. Buffy coat was
accurately removed by needle aspiration on the surface of the
pellet. The plasma supernatant was discarded and the RBC pellet was
washed three times by resuspending the RBCs in isotonic 0.9% saline
solution and centrifuging at 1600 rpm for 30 minutes at 4.degree.
C. After the final saline wash and centrifugation step, the RBC
pellet was resuspended in distilled water buffered at pH 7.2 with 5
mM potassium phosphate buffer (PB, pH=7.2) and allowed to lyse at
4.degree. C. overnight under gentle stirring. Dialyzed RBC lysate
was subsequently centrifuged at 13.000 rpm for 30 min at 4.degree.
C. and supernatant was directly loaded on an AKTA Explorer system
equipped with an XK 26/40 column packed with Q-sepharose XL resin
(GE Healthcare) at room temperature. Columns were equilibrated with
buffer A (20 mM Tris-HCl, pH=8.2) at a flow rate of 12 mL/min and
washed three times with the same buffer. A linear gradient elution
was generated by changing from 100% buffer A to 75% buffer B (20 mM
Tris-Cl, plus 0.2 M NaCl pH8.20) followed by a step gradient of
100% buffer B. Upon elution, a fraction collector was used to
collect protein fractions. Protein thus obtained was analyzed by
SDS page and stored frozen at -80.degree. C.
[0231] Human Haemoglobin (SEQ ID NO: 18 or 22, see FIG. 1) can be
readily covalently linked to appropriate drug conjugates, host
hydrophobic drug molecules within the heme binding pocket or even
transport small cytotoxic molecules linked to the heme iron. Hb can
be easily modified by selective attachment of the appropriate drug
conjugate to the cysteine residue in position 93 of the beta
chains, the only titratable cystein on the protein surface.
Maleimido functionalized drugs, such as the tubuline inhibitor
MonomethylAuristatin (MMAE) or the succinimide functionalized
mertansine analogue (DM1-SMCC) are most notable examples of
extremely potent cytotoxics that can be readily and specifically
attached to the relevant cys beta93 residue (for maleimido
functionalized drugs) or to one or more lysine residues
(succinimide functionalized drug), respectively. These drugs have
been conveniently conjugated to human haemoglobin according to the
following procedures:
[0232] The auristatin E analogue,
maleimidocaproyl-valine-citrulline-paminobenzoyloxycarbonyl-monomethyl
auristatin E (vcMMAE) was obtained from MedChem Express (Princeton,
N.J.). The Haemoglobin vcMMAE adduct was prepared as follows. Human
haemoglobin solution was adjusted to a concentration of 120 .mu.M
heme with reaction buffer (50 mM phosphate buffer pH 6,8,
containing 0.1 mM EDTA) and conjugated with 10-fold molar excess of
vcMMAE in the presence of 20% v/v acetonitrile solution at
4.degree. C. overnight. Maleimide groups react efficiently and
specifically with free (reduced) sulfhydryls at pH 6.5-7.5 to form
stable thioether bonds. The excess vcMMAE was purified and
buffer-exchanged with D-PBS using PM 100 ultrafiltration
concentrator. The yield of conjugation was approximately 80% of the
total cysteines. Formation of vcMMAE conjugate was confirmed by
LC-MS analysis and by titration of residual free thiol group with
p-chloromercuribenzoate. The concentrations of Hb-vcMMAE conjugates
were determined by UV-vis spectroscopy analysis.
[0233] The mertansin analogue DM1 SMCC (Alb Technology Ltd,
Hederson, Nev., USA), functionalized for lysine covalent
attachment, was prepared as follows. Human haemoglobin solution was
adjusted to a concentration of 400 .mu.M heme with reaction buffer
(0.1 mM phosphate buffer pH 7.4, containing 0.5 mM EDTA) and
conjugated with 20-fold molar excess of DM1-SMCC in the presence of
10% v/v DMSO solution at 4.degree. C. for 16 hours. The
amine-reactive succinimidyl ester couples to amines thus yielding a
covalent adduct with lysine groups on the surface of the protein.
The excess DM1-SMCC was eliminated and buffer-exchanged with D-PBS
using PM 100 ultrafiltration concentrator. The yield of conjugation
was approximately 2.4 mertansine molecules per haemoglobin
tetramer. Formation of DM1-SMCC conjugate was confirmed by LC-MS
analysis. The concentrations of Hb-DM1-SMCC conjugates were
determined by UV-vis spectroscopy analysis.
[0234] Alternatively, the apo-protein solution must be kept on an
ice bath for the duration of the reconstitution process. A 1.5-fold
molar excess of CuT-CPP (Cu-TCPP
4,4',4'',4'''-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic
acid)--Cu.sup.67) in 0.1 M NaOH must be added dropwise to the
apo-globin solution in 0.2 M KPi buffer at pH 7.0 vortexed quickly
at room temperature and then placed back in ice bath for 30
minutes. The protein solution must then be filtered in a syringe
filter before use.
Example 8--Preparation of Transferrin-Compound Complex
[0235] The serum was obtained from healthy donor and excess iron
was added in the presence of citrate ions as a chelator and
bicarbonate, which is facilitates for iron binding to transferrin.
The reaction mixture contained 6.5 mg sodium bicarbonate and 153.16
ferric citrate in pH=8, 4.degree. C., 1 hour per 100 mL of serum.
Albumin was subsequently precipitated by Rivanol (4%) by adding the
alcohol solution to the serum sample in a 3.5 V/V ratio at
4.degree. C., and pH=9.4 for 2 h. Then, the solution was
centrifuged at 3000 rpm for 20 min and finally filtered by filter
on a 0.8 mm syringe filter. Excess Rivanol was subsequently removed
by gel-filtration on a Sephadex G-25 column in ammonium sulfate
0.025 M. A first precipitation of by saturated ammonium sulfate 50%
at pH=6.5 was subsequently carried out followed by centrifugation
at 3000 rpm for 10 min (immunoglobulin removal). A second
precipitation at 80% saturated ammonium sulfate was then carried
out thus allowing recovery of transferrin the precipitate. Solid
precipitate was then dissolved in buffer of 0.06 M Tris HCl buffer,
pH=8, containing 1 M NaCl. The solution was dialyzed in the same
buffer to allow full removal of ammonium sulfate. Protein solution
was then concentrated with a centricon PM50 centrifugal
concentrator up to 10-15 mg/ml (as estimated by Bradford method)
and loaded on a Sephadex G-100 gel-filtration column (2,4.times.80
cm) equilibrated in 1M NaCl, flow rate of 15 ml/h. Transferrin thus
obtained was estimated to be 88-90% pure by SDS page. Ion-exchange
chromatography by anion exchanger DEAE Sephadex A-50 was then used
as a final polishing step. The transferrin sample was loaded in the
column equilibrated with 0.06 M Tris HCl at pH=8 and eluted by a
linear concentration gradient with elution buffer, 0.3 M Tris HCl,
pH=8. Protein purity was higher than 98% with a yield of about 150
mg per 100 mL of serum.
[0236] Human Holo-transferrin, (SEQ ID NO: 28, FIG. 1) similarly to
haemoglobin can be readily covalently linked to appropriate drug
conjugates, although there is only availability for lysine
modifications, due to the absence of freely titratable cysteine
groups. Thus the succinimide functionalized mertansine analogue
(DM1-SMCC) has been used to covalently attach to one or more lysine
residues (succinimide functionalized drug). The drug has been
conveniently conjugated to transferrin according to the following
procedure:
[0237] The mertansin analogue DM1 SMCC (Alb Technology Ltd,
Hederson, Nev., USA), functionalized for lysine covalent
attachment, was prepared as follows. Transferrin solution was
adjusted to a concentration of 100 .mu.M heme with reaction buffer
(0.1 mM phosphate buffer pH 7.4, no EDTA in this case due to
possible iron chelation effects) and conjugated with 20-fold molar
excess of DM1-SMCC in the presence of 8% v/v DMSO solution at
4.degree. C. for 16 hours. The amine-reactive succinimidyl ester
couples to amines thus yielding a covalent adduct with lysine
groups on the surface of the protein. The excess DM1-SMCC was
eliminated and buffer-exchanged with D-PBS using PM 100
ultrafiltration concentrator. The yield of conjugation was
approximately 1.5 mertansine molecule per transferring dimer.
Formation of DM1-SMCC conjugate was confirmed by LC-MS analysis.
The concentrations of Transferrin-DM1-SMCC conjugates were
determined by UV-vis spectroscopy analysis.
[0238] Alternatively, the apo-protein solution is kept in an ice
bath for the duration of the reconstitution process. A 1.5-fold
molar excess of CuT-CPP (Cu-TCPP
4,4',4'',4'''-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic
acid)--Cu.sup.67) in 0.1 M NaOH must be added dropwise to the
apo-globin solution in 0.2 M KPi buffer at pH 7.0 vortexed quickly
at room temperature and then placed back in ice bath for 30
minutes. The protein solution must then be filtered in a syringe
filter before use.
Example 9--Obtaining Ferritin Loaded Cells
[0239] Obtained cells are incubated in ferritin solution for a time
and at the concentration sufficient to ensure proper ratio of
ferritin/cell for their full load and also to ensure proper drug
content to obtain therapeutic effect or contrast for proper
imaging. The time and concentration may vary depending on the
number of molecules encapsulated/adsorbed into the ferritin cage,
status of cell activation, condition and number of their intended
administration.
[0240] For example, to ensure proper load with ferritins, cells are
incubated for 1-4 hours in ferritin solution 0.8 mg/ml in standard
culture conditions. The frame of ferritin concentration may vary at
least between 0.01 and 4 mg/ml as well as incubation time (5 min-6
hours or more). Adjusting time and concentration of ferritin load
to cells, the influence of ferritin and treatment conditions on
cell viability should be minded. Cells obtained as stated above
very easily uptake ferritins in a relatively short time (in
minutes; FIG. 8). Once they absorb ferritins, they do not release
it to the culture medium (FIG. 9).
[0241] Nevertheless, the person skilled in the art is able to
re-adjust the above conditions and optimize the protocol for the
own purposes in the own laboratory.
Example 10--Obtaining Haemoglobin Loaded Cells
[0242] Obtained cells are incubated in haemoglobin solution for a
time and at the concentration sufficient to ensure proper ratio of
haemoglobin/cell for their full load and also to ensure proper drug
content to obtain therapeutic effect or contrast for proper
imaging. The time and concentration may vary depending on the
number of molecules linked with the haemoglobin molecule, status of
cell activation, condition and number of their intended
administration.
[0243] For example, to ensure proper load with haemoglobins, cells
are incubated for 1-4 hours in haemoglobin solution 0.1 mg/ml in
standard culture conditions. The frame of haemoglobin concentration
may vary at least between 0.01 and 0.2 mg/ml as well as incubation
time (5 min-4 hours or more). Adjusting time and concentration of
haemoglobin load to cells, the influence of ferritin and treatment
conditions on cell viability should be minded. Cells obtained as
stated above very easily uptake haemoglobins in a relatively short
time (in minutes; FIG. 8).
[0244] Nevertheless, the person skilled in the art is able to
re-adjust the above conditions and optimize the protocol for the
own purposes in the own laboratory.
Example 11--Obtaining Transferrin Loaded Cells
[0245] Obtained cells are incubated in transferrin solution for a
time and at the concentration sufficient to ensure proper ratio of
transferrin/cell for their full load and also to ensure proper drug
content to obtain therapeutic effect or contrast for proper
imaging. The time and concentration may vary depending on the
number of molecules linked with the transferrin molecule, status of
cell activation, condition and number of their intended
administration.
[0246] For example, to ensure proper load with transferrins, cells
are incubated for 1-4 hours in transferrin solution 0.1 mg/ml in
standard culture conditions. The frame of transferrin concentration
may vary at least between 0.01 and 0.2 mg/ml as well as incubation
time (5 min-4 hours or more). Adjusting time and concentration of
transferrin load to cells, the influence of transferrin and
treatment conditions on cell viability should be minded. Cells
obtained as stated above very easily uptake transferrin in a
relatively short time (in minutes; FIG. 8).
[0247] Nevertheless, the person skilled in the art is able to
re-adjust the above conditions and optimize the protocol for the
own purposes in the own laboratory.
Example 11--Ferritin/Haemoglobin/Transferrin-Macrophage Complex as
Useful Delivery Tool to Cancer Cells
[0248] The macrophages from Example 1 prepared as described in
Examples 8, 9 and 10, very easily transport ferritins,
haemoglobins, transferrins to the cancer cells: mouse mammary
cancer, colon cancer, canine mammary cancer, human breast,
pancreatic, and bladder cancer (FIGS. 4, 10). Moreover, this
transfer is much more specific to cancer cells than to non-cancer
cells (FIG. 11). However, in case of cancer cells the ratio of both
cell types is crucial. The more macrophages the better and faster
the transport is. The most efficient transfer to the cancer cells
was observed when ratio of macrophages to cancer cells was 1:1 or
more.
[0249] This transfer occurred not only when the protein carriers
are conjugated with fluorescent label (e.g. FITC or Alexa610), but
also when they were conjugated/encapsulated with other compounds,
e.g. anticancer drugs (FIG. 12 shows this transfer of ferritin
encapsulated with fluorescent hypoxia activated
prodrug--banoxantrone). This transfer of compounds conjugated with
anticancer drugs made the effect inducing apoptosis in cancer cells
(FIG. 6, 13).
Example 12--Ferritin/Haemoglobin/Transferrin-Monocyte Complex as
Useful Delivery Tool to Cancer Cells
[0250] The monocytes from Example 2 prepared as described in
Examples 8, 9 and 10, very easily transport ferritins,
haemoglobins, transferrins to the cancer cells (FIG. 14). However,
the ratio of both cell types is important. The more monocytes the
better and faster the transport is. The most efficient transfer to
the cancer cells was observed when ratio of monocytes to cancer
cells was 1:1 or more.
Example 13--Ferritin/Haemoglobin/Transferrin-Granulocyte Complex as
Useful Delivery Tool to Cancer Cells
[0251] The granulocytes from Example 3 prepared as described in
Examples 8, 9 and 10, very easily transport ferritins,
haemoglobins, transferrins to the cancer cells (FIG. 15). However,
the ratio of both cell types is crucial. The more granulocytes the
better and faster the transport is. The most efficient transfer to
the cancer cells was observed when ratio of granulocytes to cancer
cells was 1:1 or more.
Example 14--Ferritin/Haemoglobin/Transferrin-Lymphocyte Complex as
Useful Delivery Tool to Cancer Cells
[0252] The lymphocytes from Example 4 prepared as described in
Examples 8, 9 and 10, very easily transport ferritins,
haemoglobins, transferrins to the cancer cells (FIG. 16). However,
the ratio of both cell types is crucial. The more lymphocytes the
better and faster the transport is. The most efficient transfer to
the cancer cells was observed when ratio of lymphocytes to cancer
cells was 1:1 or more.
Example 15--Cell Labeling with Radioisotope
[0253] In this invention cells were labeled with .sup.18F-FDG or
.sup.89Zr-oxinate in order to become imaged on PET. Cells have been
detached from the plate and incubated with .sup.18F-FDG or
.sup.89Zr-oxinate solution in adequate concentration to ensure the
most optimal .sup.18F-FDG or .sup.89Zr-oxinate uptake by cells
allowing their radio-detection at the site of their accumulation.
In this practice of the invention cells were incubated at room
temperature for at least 90 min. in .sup.89Zr-oxinate solution
containing 3-9 MBq per 5 min of cells. However, the ratio of
radioisotope and cells influences significantly the reaction
efficacy (FIG. 26). After labeling, cells are be centrifuged and
supernatant is removed. This step should be repeated until no
radioactivity is detected in the supernatant.
Example 16--Ferritin/Haemoglobin/Transferrin/Label-Leukocyte
Complex as Useful Delivery Tool to Tumor, Arthritic Joints and
Lymph Nodes
[0254] The macrophages from Example 1 prepared as described in
Examples 9, 10, 11 and 11; monocytes from Example 2 prepared as
described in Examples 9, 10, 11 and 12, granulocytes from Example 3
prepared as described in Examples 9, 10, 11 and 12, and lymphocytes
from Example 4 prepared as described in Examples 9, 10, 11 and 12,
highly efficiently migrate to the tumors (FIG. 22 and FIG. 23),
arthritic joints (FIG. 24), in this particular example
monocytic-macrophage cell line was used) and inflamed lymph nodes
(FIG. 25), in this particular example monocyte/young macrophages
activated using M-CSF and CCL-2 were used). Once they migrate to
the tumors or other tissues they very easily transport ferritins,
haemoglobins, transferrins and labels to the tumor in the enough
amount to be detected using imaging systems (FIGS. 2, 3, 7, 17, and
18).
Example 17--Leukocyte-Protein Carrier Complex as Useful Targeted
Pharmaceutically Active Substance Delivery System to Hypoxic
Regions
[0255] Macrophages prepared as above are injected into the tail
vein of animal with the tumour (appropriate number of macrophages
should be adjusted to the tumour size, stage of development and
presence of metastases). As it is shown on FIGS. 2, and 17 they
specifically reach the tumour (after a few hrs) and also disperse
in other organs of the whole animal (FIG. 18). Moreover, as it is
shown on FIG. 19, in hypoxic model they are also able to migrate to
the avascular and hypoxic sites and to transfer carrier proteins to
cancer cells (FIGS. 3, and 20).
[0256] For the imaging purposes, 1-50 millions of macrophages were
injected into the tail vein of mammary or colon cancer
tumour-bearing animal. Before, macrophages were pre-labeled with
Cell Tracker and loaded with ferritin-FITC (as shown in Example 8).
Using two-photon of the tumour mass 8 hours after administration of
macrophages the presence of macrophages carrying Ferritin-FITC was
detected (FIG. 17). Their specific targeting of tumour but also
their migration to other organs was shown using whole animal body
imaging (IVIS) after macrophage pre-labeling using DIR cytoplasmic
dye (FIG. 18).
[0257] The 1-10 millions of macrophages loaded with ferritin
encapsulated cyclophosphamide, melphalan and ferritin encapsulated
chlorambucil were injected i.v. into the tumour-bearing mice (300
000-500 000 of EMT6 cells injected into the skin flank). We made 3
injections of macrophages every third day (on the day 5, 8 and 11
after cancer cells injection or on the day 7, 10 and 13 after
cancer cells injection) or five consecutive injections every day
and we observed increased mouse survival (FIG. 5).
Example 18--Leukocyte-Protein Carrier Complex or Labeled Leukocyte
as Useful Targeted Label Deliver Agent
[0258] The targeting of the targeted delivery system described in
present invention can be followed by coupling the ferritin to a
contrast agent. As it is presented on FIG. 21, after injection of
1-50 ml of macrophages loaded with ferritin coupled as described in
Example 8 with a contrast agent (in this case: ferrihydrite,
however the same results are obtained with isotope, e.g. .sup.123I)
or labeled with isotope (in this case .sup.89Zr-oxide or
.sup.18F-FDG) (FIG. 21) they can be easily detected by MRI, PET or
SPECT. In this example (FIG. 7), mammary-tumour bearing mice were
imaged using MRI at 3, 22 and 24 hours after i.v. injection of
macrophages loaded with ferritin Fh. The mouse was treated (at time
point 0 h) with macrophages. Then increased diameter of blood
vessels (arrow) filled with injected macrophages (giving
significant T2-signal reduction) has been observed and afterword
macrophages spread to the tissue (spot-like pattern; arrows). These
changes (in the same time points) were observed in all examined
mice.
[0259] Macrophages were also labeled with .sup.89Zr-oxide or
.sup.18F-FDG (5-50 min) and their radioactivity was confirmed to be
imaged using PET after i.v. administration to the tumour-bearing
mice. These mice were inoculated with 4T1 metastatic cell line 3
weeks before the experiment and metastases in the lungs, liver and
spleen were histopatologically confirmed. At FIG. 21 it is seen
that macrophages migrated to the regions with metastatic tumours
allowing their visualization at PET.
[0260] While the foregoing written description of the invention
enables one of ordinary skill to make and use what is considered
presently to be the best mode thereof, those of ordinary skill will
understand and appreciate the existence of variations,
combinations, and equivalents of the specific embodiment, method,
and examples herein. The invention should therefore not be limited
by the above described embodiment, method, and examples, but by all
embodiments and methods within the scope and spirit of the
invention.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 28 <210> SEQ ID NO 1 <211> LENGTH: 132 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Minimal consensus sequence
of mammalian ferritin H chain <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa at position 5 may be present or absent, if
present it means any naturally occurring amino acid, preferably Ile
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa at position 6
means any naturally occurring amino acid, preferably Asn
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: Xaa at position
14 means any naturally occurring amino acid, preferably Tyr
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION: Xaa at position
24 means any naturally occurring amino acid, preferably Tyr or Cys,
more preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (66)..(66) <223> OTHER
INFORMATION: Xaa at position 66 means any naturally occurring amino
acid, preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa at position 68 means any naturally occurring amino
acid, preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (75)..(75) <223> OTHER
INFORMATION: Xaa at position 75 means any naturally occurring amino
acid, preferably Arg or Cys, more preferably Cys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(90)..(90) <223> OTHER INFORMATION: Xaa at position 90 means
any naturally occurring amino acid, preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(94)..(94) <223> OTHER INFORMATION: Xaa at position 94 means
any naturally occurring amino acid, preferably Ser or Asn, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (120)..(120) <223> OTHER
INFORMATION: Xaa at position 120 may be present or absent, if
present it means any naturally occurring amino acid, preferably His
or Tyr, more preferably His <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (123)..(123)
<223> OTHER INFORMATION: Xaa at position 123 means any
naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (128)..(128) <223> OTHER
INFORMATION: Xaa at position 128 means any naturally occurring
amino acid, preferably Ala or Ser, more preferably Ala <400>
SEQUENCE: 1 Ser Glu Ala Ala Xaa Xaa Arg Gln Ile Asn Leu Glu Leu Xaa
Ala Ser 1 5 10 15 Tyr Val Tyr Leu Ser Met Ser Xaa Tyr Phe Asp Arg
Asp Asp Val Ala 20 25 30 Leu Lys Asn Phe Ala Lys Tyr Phe Leu His
Gln Ser His Glu Glu Arg 35 40 45 Glu His Ala Glu Lys Leu Met Lys
Leu Gln Asn Gln Arg Gly Gly Arg 50 55 60 Ile Xaa Leu Xaa Asp Ile
Lys Lys Pro Asp Xaa Asp Asp Trp Glu Ser 65 70 75 80 Gly Leu Asn Ala
Met Glu Cys Ala Leu Xaa Leu Glu Lys Xaa Asn Gln 85 90 95 Ser Leu
Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His 100 105 110
Leu Cys Asp Phe Ile Glu Thr Xaa Tyr Leu Xaa Glu Gln Val Lys Xaa 115
120 125 Ile Lys Glu Leu 130 <210> SEQ ID NO 2 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Consensus sequence of mammalian ferritin H chain <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(6)..(6) <223> OTHER INFORMATION: Xaa at position 6 can be
any naturally occurring amino acid, preferably Pro <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(14)..(14) <223> OTHER INFORMATION: Xaa at position 14 can be
any naturally occurring amino acid, preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(16)..(16) <223> OTHER INFORMATION: Xaa at position 16 can be
any naturally occurring amino acid, preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Xaa at position 21 may be
present or absent, if present it means any naturally occurring
amino acid, preferably Ile <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (22)..(22) <223>
OTHER INFORMATION: Xaa at position 22 means any naturally occurring
amino acid, preferably Asn <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (30)..(30) <223>
OTHER INFORMATION: Xaa at position 30 can be any naturally
occurring amino acid, preferably Tyr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (40)..(40)
<223> OTHER INFORMATION: Xaa at position 40 can be any
naturally occurring amino acid, preferably Tyr or Cys, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (82)..(82) <223> OTHER
INFORMATION: Xaa at position 82 can be any naturally occurring
amino acid, preferably Phe <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (84)..(84) <223>
OTHER INFORMATION: Xaa at position 84 can be any naturally
occurring amino acid, preferably Gln <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (91)..(91)
<223> OTHER INFORMATION: Xaa at position 91 can be any
naturally occurring amino acid, preferably Arg or Cys, more
preferably Cys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (106)..(106) <223> OTHER
INFORMATION: Xaa at position 106 can be any naturally occurring
amino acid, preferably His <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (110)..(110)
<223> OTHER INFORMATION: Xaa at position 110 can be any
naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (137)..(137) <223> OTHER
INFORMATION: Xaa at position 137 can be any naturally occurring
amino acid, preferably His or Tyr, more preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(140)..(140) <223> OTHER INFORMATION: Xaa at position 140 can
be any naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (145)..(145) <223> OTHER
INFORMATION: Xaa at position 145 can be any naturally occurring
amino acid, preferably Ala or Ser, more preferably Ala <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(164)..(164) <223> OTHER INFORMATION: Xaa at position 164 can
be any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (166)..(166) <223> OTHER
INFORMATION: Xaa at position 166 can be any naturally occurring
amino acid, preferably Met or Leu, prerferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(178)..(178) <223> OTHER INFORMATION: Xaa at position 178 can
be any naturally occurring amino acid, preferably Asp or His, more
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (181)..(181) <223> OTHER
INFORMATION: Xaa at position 181 is absent or any naturally
occurring amino acid, preferably Asn <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(182)..(182) <223> OTHER INFORMATION: Xaa at position 182 is
absent or any naturally occurring amino acid, preferably Glu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (183)..(183) <223> OTHER INFORMATION: Xaa at
position 183 is absent or any naturally occurring amino acid,
preferably Ser <400> SEQUENCE: 2 Met Thr Thr Ala Ser Xaa Ser
Gln Val Arg Gln Asn Tyr Xaa Gln Xaa 1 5 10 15 Ser Glu Ala Ala Xaa
Xaa Arg Gln Ile Asn Leu Glu Leu Xaa Ala Ser 20 25 30 Tyr Val Tyr
Leu Ser Met Ser Xaa Tyr Phe Asp Arg Asp Asp Val Ala 35 40 45 Leu
Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg 50 55
60 Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg
65 70 75 80 Ile Xaa Leu Xaa Asp Ile Lys Lys Pro Asp Xaa Asp Asp Trp
Glu Ser 85 90 95 Gly Leu Asn Ala Met Glu Cys Ala Leu Xaa Leu Glu
Lys Xaa Val Asn 100 105 110 Gln Ser Leu Leu Glu Leu His Lys Leu Ala
Thr Asp Lys Asn Asp Pro 115 120 125 His Leu Cys Asp Phe Ile Glu Thr
Xaa Tyr Leu Xaa Glu Gln Val Lys 130 135 140 Xaa Ile Lys Glu Leu Gly
Asp His Val Thr Asn Leu Arg Lys Met Gly 145 150 155 160 Ala Pro Glu
Xaa Gly Xaa Ala Glu Tyr Leu Phe Asp Lys His Thr Leu 165 170 175 Gly
Xaa Ser Asp Xaa Xaa Xaa 180 <210> SEQ ID NO 3 <211>
LENGTH: 133 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 3 Ala Glu Ala Ala Ile Asn Arg Gln
Ile Asn Leu Glu Leu Tyr Ala Ser 1 5 10 15 Tyr Val Tyr Leu Ser Met
Ser Cys Tyr Phe Asp Arg Asp Asp Val Ala 20 25 30 Leu Lys Asn Phe
Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg 35 40 45 Glu His
Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg 50 55 60
Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp Asp Trp Glu Ser 65
70 75 80 Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Ser
Val Asn 85 90 95 Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp
Lys Asn Asp Pro 100 105 110 His Leu Cys Asp Phe Ile Glu Thr Tyr Tyr
Leu Ser Glu Gln Val Lys 115 120 125 Ser Ile Lys Glu Leu 130
<210> SEQ ID NO 4 <211> LENGTH: 180 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 4 Met
Thr Thr Ala Ser Pro Ser Gln Val Arg Gln Asn Tyr His Gln Asp 1 5 10
15 Ala Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser
20 25 30 Tyr Val Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp
Val Ala 35 40 45 Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser
His Glu Glu Arg 50 55 60 Glu His Ala Glu Lys Leu Met Lys Leu Gln
Asn Gln Arg Gly Gly Arg 65 70 75 80 Ile Phe Leu Gln Asp Ile Lys Lys
Pro Asp Arg Asp Asp Trp Glu Ser 85 90 95 Gly Leu Asn Ala Met Glu
Cys Ala Leu His Leu Glu Lys Ser Val Asn 100 105 110 Gln Ser Leu Leu
Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro 115 120 125 His Leu
Cys Asp Phe Ile Glu Thr Tyr Tyr Leu Ser Glu Gln Val Lys 130 135 140
Ser Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly 145
150 155 160 Ala Pro Glu Ala Gly Met Ala Glu Tyr Leu Phe Asp Lys His
Thr Leu 165 170 175 Gly His Gly Asp 180 <210> SEQ ID NO 5
<211> LENGTH: 132 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 5 Ser Glu Ala Ala Asn Arg Gln
Ile Asn Leu Glu Leu Tyr Ala Ser Tyr 1 5 10 15 Val Tyr Leu Ser Met
Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu 20 25 30 Lys Asn Phe
Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu 35 40 45 His
Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile 50 55
60 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly
65 70 75 80 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val
Asn Gln 85 90 95 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys
Asn Asp Pro His 100 105 110 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu
Asn Glu Gln Val Lys Ala 115 120 125 Ile Lys Glu Leu 130 <210>
SEQ ID NO 6 <211> LENGTH: 179 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 Met Thr
Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp 1 5 10 15
Ser Glu Ala Ala Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr 20
25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala
Leu 35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu
Glu Arg Glu 50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln
Arg Gly Gly Arg Ile 65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp
Cys Asp Asp Trp Glu Ser Gly 85 90 95 Leu Asn Ala Met Glu Cys Ala
Leu His Leu Glu Lys Asn Val Asn Gln 100 105 110 Ser Leu Leu Glu Leu
His Lys Leu Ala Thr Asp Lys Asn Asp Pro His 115 120 125 Leu Cys Asp
Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala 130 135 140 Ile
Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150
155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu
Gly 165 170 175 Asp Ser Asp <210> SEQ ID NO 7 <211>
LENGTH: 182 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Consensus sequence of mammalian ferritin H chains 2nd version
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa at position 6
can be any naturally occurring amino acid, preferably Pro
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: Xaa at position
14 can be any naturally occurring amino acid, preferably His
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (16)..(16) <223> OTHER INFORMATION: Xaa at position
16 can be any naturally occurring amino acid, preferably Asp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)..(21) <223> OTHER INFORMATION: Xaa at position
21 may be present or absent, if present it means any naturally
occurring amino acid, preferably Ile <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (29)..(29)
<223> OTHER INFORMATION: Xaa at position 29 can be any
naturally occurring amino acid, preferably Tyr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (81)..(81)
<223> OTHER INFORMATION: Xaa at position 81 can be any
naturally occurring amino acid, preferably Phe <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (83)..(83)
<223> OTHER INFORMATION: Xaa at position 83 can be any
naturally occurring amino acid, preferably Gln <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(105)..(105) <223> OTHER INFORMATION: Xaa at position 105 can
be any naturally occurring amino acid, preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(144)..(144) <223> OTHER INFORMATION: Xaa at position 144 can
be any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (180)..(180) <223> OTHER
INFORMATION: Xaa at position 180 is absent or any naturally
occuring amino acid, preferably Asn <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(181)..(181) <223> OTHER INFORMATION: Xaa at position 181 is
absent or any naturally occuring amino acid, preferably Glu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (182)..(182) <223> OTHER INFORMATION: Xaa at
position 182 is absent or any naturally occuring amino acid,
preferably Ser <400> SEQUENCE: 7 Met Thr Thr Ala Ser Xaa Ser
Gln Val Arg Gln Asn Tyr Xaa Gln Xaa 1 5 10 15 Ser Glu Ala Ala Xaa
Arg Gln Ile Asn Leu Glu Leu Xaa Ala Ser Tyr 20 25 30 Val Tyr Leu
Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu 35 40 45 Lys
Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu 50 55
60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile
65 70 75 80 Xaa Leu Xaa Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu
Ser Gly 85 90 95 Leu Asn Ala Met Glu Cys Ala Leu Xaa Leu Glu Lys
Asn Val Asn Gln 100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr
Asp Lys Asn Asp Pro His 115 120 125 Leu Cys Asp Phe Ile Glu Thr His
Tyr Leu Asn Glu Gln Val Lys Xaa 130 135 140 Ile Lys Glu Leu Gly Asp
His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser
Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly 165 170 175 Asp
Ser Asp Xaa Xaa Xaa 180 <210> SEQ ID NO 8 <211> LENGTH:
150 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Minimal
consensus sequence of mammalian ferritin L chain <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Xaa at position 5 can be
any naturally occurring amino acid, preferably Asp or Glu, more
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa at position 12 can be any nnaturally occurring
amino acid, preferably Arg or Ser, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(17)..(17) <223> OTHER INFORMATION: Xaa at position 17 can be
any naturally occurring amino acid, preferably Ser or Arg, more
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa at position 19 can be any naturally occurring
amino acid, preferably Arg or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(29)..(29) <223> OTHER INFORMATION: Xaa at position 29 can be
any naturally occurring amino acid, preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(30)..(30) <223> OTHER INFORMATION: Xaa at position 30 can be
any naturally occurring amino acid, preferably Tyr or Phe, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: Xaa at position 42 can be any naturally occurring
amino acid, preferably Ser or Gly, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(56)..(56) <223> OTHER INFORMATION: Xaa at position 56 can be
any naturally occurirng amino acid, preferably Ala or Tyr, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (61)..(61) <223> OTHER
INFORMATION: Xaa at position 61 can be any naturally occurring
amino acid, preferably Glu or Lys, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(62)..(62) <223> OTHER INFORMATION: Xaa at position 62 can be
any naturally occurring amino acid, preferably Met or Phe, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa at position 65 can be any naturally occurring
amino acid, preferably Asp or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(75)..(75) <223> OTHER INFORMATION: Xaa at position 75 can be
any naturally occurring amino acid, preferably Ile or Val, more
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (76)..(76) <223> OTHER
INFORMATION: Xaa at position 76 can be any naturally occurring
amino acid, preferably Lys or Gln, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(79)..(79) <223> OTHER INFORMATION: Xaa at position 79 can be
any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (80)..(80) <223> OTHER
INFORMATION: Xaa at position 80 can be any naturally occurring
amino acid, preferably Glu or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(87)..(87) <223> OTHER INFORMATION: Xaa at position 87 can be
any naturally occurring amino acid, preferably Pro or Gln, more
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (88)..(88) <223> OTHER
INFORMATION: Xaa at position 88 can be any naturally occurring
amino acid, preferably Glu or Asp, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(91)..(91) <223> OTHER INFORMATION: Xaa at position 91 can be
any naturally occurring amino acid, preferably Glu or Lys, more
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa at position 94 can be any naturally occurring
amino acid, preferably Met or Leu, more preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(96)..(96) <223> OTHER INFORMATION: Xaa at position 96 can be
any naturally occurring amino acid, preferably Met or Leu, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (99)..(99) <223> OTHER
INFORMATION: Xaa at position 99 can be any naturally occurring
amino acid, preferably Lys or Asn, preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(115)..(115) <223> OTHER INFORMATION: Xaa at position 115 can
be any naturally occurring amino acid, preferably Thr or Ala, more
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (119)..(119) <223> OTHER
INFORMATION: Xaa at position 119 can be any naturally occurring
amino acid, preferably Leu <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (125)..(125)
<223> OTHER INFORMATION: Xaa at position 125 can be any
naturally occurring amino acid, preferably Ser or Thr, more
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (127)..(127) <223> OTHER
INFORMATION: Xaa at position 127 can be any naturally occurring
amino acid, preferably Tyr or Phe, more preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(130)..(130) <223> OTHER INFORMATION: Xaa at position 130 can
be any naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (140)..(140) <223> OTHER
INFORMATION: Xaa at position 140 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(146)..(146) <223> OTHER INFORMATION: Xaa at position 146 can
be any naturally occurring amino acid, preferably Arg or His, more
preferably His <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (148)..(148) <223> OTHER
INFORMATION: Xaa at position 148 can be any naturally occurring
amino acid, preferably Leu or Val, more preferably Leu <400>
SEQUENCE: 8 Asn Tyr Ser Thr Xaa Val Glu Ala Ala Val Asn Xaa Leu Val
Asn Leu 1 5 10 15 Xaa Leu Xaa Ala Ser Tyr Thr Tyr Leu Ser Leu Gly
Xaa Xaa Phe Asp 20 25 30 Arg Asp Asp Val Ala Leu Glu Gly Val Xaa
His Phe Phe Arg Glu Leu 35 40 45 Ala Glu Glu Lys Arg Glu Gly Xaa
Glu Arg Leu Leu Xaa Xaa Gln Asn 50 55 60 Xaa Arg Gly Gly Arg Ala
Leu Phe Gln Asp Xaa Xaa Lys Pro Xaa Xaa 65 70 75 80 Asp Glu Trp Gly
Lys Thr Xaa Xaa Ala Met Xaa Ala Ala Xaa Ala Xaa 85 90 95 Glu Lys
Xaa Leu Asn Gln Ala Leu Leu Asp Leu His Ala Leu Gly Ser 100 105 110
Ala Arg Xaa Asp Pro His Xaa Cys Asp Phe Leu Glu Xaa His Xaa Leu 115
120 125 Asp Xaa Glu Val Lys Leu Ile Lys Lys Met Gly Xaa His Leu Thr
Asn 130 135 140 Leu Xaa Arg Xaa Ala Gly 145 150 <210> SEQ ID
NO 9 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus sequence of mammalian ferritin L chain
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa at position
12 can be any naturally occurring amino acid, preferably Asp or
Glu, more preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa at position 19 can be any naturally occurring
amino acid, preferably Ser or Arg, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa at position 24 can be
any naturally occurring amino acid, preferably Ser or Arg, more
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa at position 26 can be any naturally occurring
amino acid, preferably Arg or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(36)..(36) <223> OTHER INFORMATION: Xaa at position 36 can be
any naturally occurring amino acid, preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(37)..(37) <223> OTHER INFORMATION: Xaa at position 37 can be
any naturally occurring amino acid, preferably Tyr or Phe, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (49)..(49) <223> OTHER
INFORMATION: Xaa at position 47 can be any naturally occurring
amino acid, preferably Ser or Gly, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(63)..(63) <223> OTHER INFORMATION: Xaa at position 63 can be
any naturally occurring amino acid, preferably Ala or Tyr, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa at position 68 can be any naturally occurring
amino acid, preferably Glu or Lys, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(69)..(69) <223> OTHER INFORMATION: Xaa at position 69 can be
any naturally occurring amino acid, preferably Met or Phe, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (72)..(72) <223> OTHER
INFORMATION: Xaa at position 72 can be any naturally occurring
amino acid, preferably Asp or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(82)..(82) <223> OTHER INFORMATION: Xaa at position 82 can be
any naturally occurring amino acid, preferably Ile or Val, more
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (83)..(83) <223> OTHER
INFORMATION: Xaa at position 83 can be any naturally occurring
amino acid, preferably Lys or Gln, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(86)..(86) <223> OTHER INFORMATION: Xaa at position 86 can be
any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (87)..(87) <223> OTHER
INFORMATION: Xaa at position 87 can be any naturally occurring
amino acid, preferably Glu or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(94)..(94) <223> OTHER INFORMATION: Xaa at position 94 can be
any naturally occurring amino acid, preferably Pro or Gln, more
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (95)..(95) <223> OTHER
INFORMATION: Xaa at position 95 can be any naturally occurring
amino acid, preferably Glu or Asp, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(98)..(98) <223> OTHER INFORMATION: Xaa at position 98 can be
any naturally occurring amino acid, preferably Glu or Lys, more
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (101)..(101) <223> OTHER
INFORMATION: Xaa at position 101 can be any naturally occurring
amino acid, preferably Met or Leu, more preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(103)..(103) <223> OTHER INFORMATION: Xaa at position 103 can
be any naturally occurring amino acid, preferably Met or Leu, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (106)..(106) <223> OTHER
INFORMATION: Xaa at position 106 can be any naturally occurring
amino acid, preferably Lys or Asn, preferably Lys <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(122)..(122) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (126)..(126)
<223> OTHER INFORMATION: Xaa at position 126 can be any
naturally occurring amino acid, preferably Leu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(132)..(132) <223> OTHER INFORMATION: Xaa at position 132 can
be any naturally occurring amino acid, preferably Ser or Thr, more
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (134)..(134) <223> OTHER
INFORMATION: Xaa at position 134 can be any naturally occurring
amino acid, preferably Tyr or Phe, more preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(137)..(137) <223> OTHER INFORMATION: Xaa at position 137 can
be any naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (147)..(147) <223> OTHER
INFORMATION: Xaa at position 147 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(153)..(153) <223> OTHER INFORMATION: Xaa at position 153 can
be any naturally occurring amino acid, preferably Arg or His, more
preferably His <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (155)..(155) <223> OTHER
INFORMATION: Xaa at position 155 can be any naturally occurring
amino acid, preferably Leu or Val, more preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(161)..(161) <223> OTHER INFORMATION: Xaa at position 161 can
be absent or any naturally occurring amino acid, preferably Ala
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (163)..(163) <223> OTHER INFORMATION: Xaa at
position 163 can be absent or any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (166)..(166) <223> OTHER
INFORMATION: Xaa at position 166 can be absent or any naturally
occurring amino acid, preferably Pro <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(168)..(168) <223> OTHER INFORMATION: Xaa at position 168 can
be any naturally occurring amino acid, preferably Gly or Ala, more
preferably Ala <400> SEQUENCE: 9 Met Thr Ser Gln Ile Arg Gln
Asn Tyr Ser Thr Xaa Val Glu Ala Ala 1 5 10 15 Val Asn Xaa Leu Val
Asn Leu Xaa Leu Xaa Ala Ser Tyr Thr Tyr Leu 20 25 30 Ser Leu Gly
Xaa Xaa Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val 35 40 45 Xaa
His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Xaa Glu 50 55
60 Arg Leu Leu Xaa Xaa Gln Asn Xaa Arg Gly Gly Arg Ala Leu Phe Gln
65 70 75 80 Asp Xaa Xaa Lys Pro Xaa Xaa Asp Glu Trp Gly Lys Thr Xaa
Xaa Ala 85 90 95 Met Xaa Ala Ala Xaa Ala Xaa Glu Lys Xaa Leu Asn
Gln Ala Leu Leu 100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Xaa
Asp Pro His Xaa Cys Asp 115 120 125 Phe Leu Glu Xaa His Xaa Leu Asp
Xaa Glu Val Lys Leu Ile Lys Lys 130 135 140 Met Gly Xaa His Leu Thr
Asn Leu Xaa Arg Xaa Ala Gly Pro Gln Pro 145 150 155 160 Xaa Gln Xaa
Gly Val Xaa Gln Xaa Ser Leu Gly Glu Tyr Leu Phe Glu 165 170 175 Arg
Leu Thr Leu Lys His Asp 180 <210> SEQ ID NO 10 <211>
LENGTH: 150 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
at position 5 can be any naturally occuring amino acid, preferably
Asp or Glu, more preferably Asp <400> SEQUENCE: 10 Asn Tyr
Ser Thr Glu Val Glu Ala Ala Val Asn Arg Leu Val Asn Leu 1 5 10 15
His Leu Arg Ala Ser Tyr Thr Tyr Leu Ser Leu Gly Phe Phe Phe Asp 20
25 30 Arg Asp Asp Val Ala Leu Glu Gly Val Gly His Phe Phe Arg Glu
Leu 35 40 45 Ala Glu Glu Lys Arg Glu Gly Ala Glu Arg Leu Leu Glu
Phe Gln Asn 50 55 60 Asp Arg Gly Gly Arg Ala Leu Phe Gln Asp Val
Gln Lys Pro Ser Gln 65 70 75 80 Asp Glu Trp Gly Lys Thr Gln Glu Ala
Met Glu Ala Ala Leu Ala Met 85 90 95 Glu Lys Asn Leu Asn Gln Ala
Leu Leu Asp Leu His Ala Leu Gly Ser 100 105 110 Ala Arg Ala Asp Pro
His Leu Cys Asp Phe Leu Glu Ser His Tyr Leu 115 120 125 Asp Lys Glu
Val Lys Leu Ile Lys Lys Met Gly Asn His Leu Thr Asn 130 135 140 Leu
Arg Arg Val Ala Gly 145 150 <210> SEQ ID NO 11 <400>
SEQUENCE: 11 000 <210> SEQ ID NO 12 <211> LENGTH: 149
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Asn Tyr Ser Thr Asp Val Glu Ala Ala Val
Asn Ser Leu Val Asn Leu 1 5 10 15 Tyr Leu Gln Ala Ser Tyr Thr Tyr
Leu Ser Leu Gly Phe Tyr Phe Asp 20 25 30 Arg Asp Asp Val Ala Leu
Glu Gly Val Ser His Phe Phe Arg Glu Leu 35 40 45 Ala Glu Glu Lys
Arg Glu Gly Tyr Glu Arg Leu Leu Lys Met Gln Asn 50 55 60 Gln Arg
Gly Gly Arg Ala Leu Phe Gln Asp Ile Lys Lys Pro Ala Glu 65 70 75 80
Asp Glu Trp Gly Lys Thr Pro Asp Ala Met Lys Ala Ala Met Ala Leu 85
90 95 Glu Lys Lys Leu Asn Gln Ala Leu Leu Asp Leu His Ala Leu Gly
Ser 100 105 110 Ala Arg Thr Asp Pro His Leu Cys Asp Phe Leu Glu Thr
His Phe Leu 115 120 125 Asp Glu Glu Val Lys Leu Ile Lys Lys Met Gly
Asp His Leu Thr Asn 130 135 140 Leu His Arg Leu Gly 145 <210>
SEQ ID NO 13 <211> LENGTH: 175 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 Met Ser
Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala 1 5 10 15
Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu 20
25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly
Val 35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu
Gly Tyr Glu 50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly
Arg Ala Leu Phe Gln 65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu
Trp Gly Lys Thr Pro Asp Ala 85 90 95 Met Lys Ala Ala Met Ala Leu
Glu Lys Lys Leu Asn Gln Ala Leu Leu 100 105 110 Asp Leu His Ala Leu
Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp 115 120 125 Phe Leu Glu
Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys 130 135 140 Met
Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145 150
155 160 Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu Thr Leu Lys His Asp
165 170 175 <210> SEQ ID NO 14 <211> LENGTH: 183
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
seqeuence of mammaliam ferritin L chain 2nd version <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(36)..(36) <223> OTHER INFORMATION: Xaa at position 36 can be
any naturally occuring amino acid, preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(37)..(37) <223> OTHER INFORMATION: Xaa at position 37 can be
any naturally occuring amino acid, preferably Tyr or Phe, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa at position 94 can be any naturally occurring
amino acid, preferably Pro or Gln, more preferably Pro <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(126)..(126) <223> OTHER INFORMATION: Xaa at position 126 can
be any naturally occurring amino acid, preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(137)..(137) <223> OTHER INFORMATION: Xaa at position 137 can
be any naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (147)..(147) <223> OTHER
INFORMATION: Xaa at position 147 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(161)..(161) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (163)..(163)
<223> OTHER INFORMATION: Xaa at position 163 can be absent or
any naturally occurring amino acid, preferably Thr <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(166)..(166) <223> OTHER INFORMATION: Xaa at position 166 can
be absent or any naturally occurring amino acid, preferably Pro
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (168)..(168) <223> OTHER INFORMATION: Xaa at
position 168 can be any naturally occurring amino acid, preferably
Gly or Ala, more preferably Ala <400> SEQUENCE: 14 Met Thr
Ser Gln Ile Arg Gln Asn Tyr Ser Thr Glu Val Glu Ala Ala 1 5 10 15
Val Asn Arg Leu Val Asn Leu His Leu Arg Ala Ser Tyr Thr Tyr Leu 20
25 30 Ser Leu Gly Xaa Xaa Phe Asp Arg Asp Asp Val Ala Leu Glu Gly
Val 35 40 45 Gly His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu
Gly Ala Glu 50 55 60 Arg Leu Leu Lys Leu Gln Asn Gln Arg Gly Gly
Arg Ala Leu Phe Gln 65 70 75 80 Asp Val Gln Lys Pro Ala Gln Asp Glu
Trp Gly Lys Thr Xaa Glu Ala 85 90 95 Met Glu Ala Ala Leu Ala Leu
Glu Lys Asn Leu Asn Gln Ala Leu Leu 100 105 110 Asp Leu His Ala Leu
Gly Ser Ala Arg Thr Asp Pro His Xaa Cys Asp 115 120 125 Phe Leu Glu
Asn His Phe Leu Asp Xaa Glu Val Lys Leu Ile Lys Lys 130 135 140 Met
Gly Xaa His Leu Thr Asn Leu Arg Arg Leu Ala Gly Pro Gln Pro 145 150
155 160 Xaa Gln Xaa Gly Val Xaa Gln Xaa Ser Leu Gly Glu Tyr Leu Phe
Glu 165 170 175 Arg Leu Thr Leu Lys His Asp 180 <210> SEQ ID
NO 15 <211> LENGTH: 117 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Minimal consensus sequence of mammalian
haemoglobin alpha chain <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (41)..(41) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (48)..(48) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (49)..(49) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (55)..(55) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (59)..(59) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Aap <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (60)..(60) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (76)..(76) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (79)..(79) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (90)..(90) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (91)..(91) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (102)..(102) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (117)..(117) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <400> SEQUENCE: 15 Val Gly Ala His Xaa Gly Glu
Tyr Xaa Ala Glu Ala Leu Glu Xaa Met 1 5 10 15 Phe Leu Ser Phe Xaa
Xaa Thr Lys Thr Tyr Phe Pro His Phe Asp Leu 20 25 30 Ser His Gly
Ser Ala Xaa Val Lys Xaa His Gly Lys Lys Val Ala Xaa 35 40 45 Xaa
Leu Thr Asn Ala Val Xaa His Val Asp Xaa Xaa Pro Asn Ala Leu 50 55
60 Ser Ala Leu Xaa Asp Leu His Ala His Lys Leu Xaa Val Asp Xaa Val
65 70 75 80 Asn Phe Lys Leu Leu Ser His Cys Leu Xaa Xaa Thr Leu Xaa
Ala His 85 90 95 Leu Pro Ala Glu Phe Xaa Pro Ala Val His Ala Xaa
Leu Asp Lys Phe 100 105 110 Leu Ala Ser Val Xaa 115 <210> SEQ
ID NO 16 <211> LENGTH: 142 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus sequence of mammalian alpha chain
haemoglobins <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (17)..(17) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (32)..(32) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (39)..(39) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (55)..(55) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (58)..(58) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (66)..(66) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (72)..(72) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (76)..(76) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (77)..(77) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (85)..(85) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (93)..(93) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (96)..(96) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (107)..(107) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (111)..(111) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (119)..(119) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (125)..(125) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (134)..(134) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (142)..(142) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <400> SEQUENCE: 16 Xaa Xaa Leu Ser Pro Ala Asp
Lys Thr Asn Val Lys Ala Xaa Trp Gly 1 5 10 15 Xaa Val Gly Ala His
Xaa Gly Glu Tyr Xaa Ala Glu Ala Leu Glu Xaa 20 25 30 Met Phe Leu
Ser Phe Xaa Xaa Thr Lys Thr Tyr Phe Pro His Phe Asp 35 40 45 Leu
Ser His Gly Ser Ala Xaa Val Lys Xaa His Gly Lys Lys Val Ala 50 55
60 Xaa Xaa Leu Thr Asn Ala Val Xaa His Val Asp Xaa Xaa Pro Asn Ala
65 70 75 80 Leu Ser Ala Leu Xaa Asp Leu His Ala His Lys Leu Xaa Val
Asp Xaa 85 90 95 Val Asn Phe Lys Leu Leu Ser His Cys Leu Xaa Xaa
Thr Leu Xaa Ala 100 105 110 His Leu Pro Ala Glu Phe Xaa Pro Ala Val
His Ala Xaa Leu Asp Lys 115 120 125 Phe Leu Ala Ser Val Xaa Thr Val
Leu Thr Ser Lys Tyr Xaa 130 135 140 <210> SEQ ID NO 17
<211> LENGTH: 117 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 17 Val Gly Ala His Ala Gly Glu
Tyr Gly Ala Glu Ala Leu Glu Arg Met 1 5 10 15 Phe Leu Ser Phe Pro
Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu 20 25 30 Ser His Gly
Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala Asp 35 40 45 Ala
Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala Leu 50 55
60 Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro Val
65 70 75 80 Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala
Ala His 85 90 95 Leu Pro Ala Glu Phe Thr Pro Ala Val His Ala Ser
Leu Asp Lys Phe 100 105 110 Leu Ala Ser Val Ser 115 <210> SEQ
ID NO 18 <211> LENGTH: 142 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 Met Val Leu Ser Pro
Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly 1 5 10 15 Lys Val Gly
Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg 20 25 30 Met
Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp 35 40
45 Leu Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala
50 55 60 Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro
Asn Ala 65 70 75 80 Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu
Arg Val Asp Pro 85 90 95 Val Asn Phe Lys Leu Leu Ser His Cys Leu
Leu Val Thr Leu Ala Ala 100 105 110 His Leu Pro Ala Glu Phe Thr Pro
Ala Val His Ala Ser Leu Asp Lys 115 120 125 Phe Leu Ala Ser Val Ser
Thr Val Leu Thr Ser Lys Tyr Arg 130 135 140 <210> SEQ ID NO
19 <211> LENGTH: 124 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Minimal consensus sequence of mammalian
haemoglobin beta chain <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (36)..(36) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (49)..(49) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (52)..(52) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (66)..(66) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (69)..(69) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (86)..(86) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Glu <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (121)..(121) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (122)..(122) <223> OTHER INFORMATION: Xaa can be
absent or any naturally occurring amino acid, preferably Val
<400> SEQUENCE: 19 Gly Lys Val Xaa Val Asp Glu Val Xaa Xaa
Glu Xaa Xaa Xaa Xaa Leu 1 5 10 15 Leu Val Val Tyr Pro Trp Thr Gln
Arg Phe Xaa Glu Ser Phe Gly Asp 20 25 30 Leu Ser Xaa Xaa Asp Ala
Val Met Gly Asn Pro Lys Val Lys Ala His 35 40 45 Xaa Lys Lys Xaa
Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp 50 55 60 Asn Xaa
Lys Xaa Xaa Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys 65 70 75 80
Leu His Val Asp Pro Xaa Asn Phe Arg Leu Leu Gly Asn Val Leu Val 85
90 95 Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val
Gln 100 105 110 Ala Ala Tyr Gln Lys Val Val Ala Xaa Xaa Ala Asn 115
120 <210> SEQ ID NO 20 <211> LENGTH: 147 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Consensus sequence of
mammalian haemoglobin beta chain <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (20)..(20) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (28)..(28) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (29)..(29) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (30)..(30) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (31)..(31) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (43)..(43) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (51)..(51) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (52)..(52) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (82)..(82) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (84)..(84) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (85)..(85) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (102)..(102) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (137)..(137) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (138)..(138) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (147)..(147) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably His <400> SEQUENCE: 20 Met Val His Leu Thr Pro Glu
Glu Lys Xaa Xaa Val Thr Ala Xaa Trp 1 5 10 15 Gly Lys Val Xaa Val
Asp Glu Val Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu 20 25 30 Leu Val Val
Tyr Pro Trp Thr Gln Arg Phe Xaa Glu Ser Phe Gly Asp 35 40 45 Leu
Ser Xaa Xaa Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His 50 55
60 Xaa Lys Lys Xaa Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp
65 70 75 80 Asn Xaa Lys Xaa Xaa Phe Ala Thr Leu Ser Glu Leu His Cys
Asp Lys 85 90 95 Leu His Val Asp Pro Xaa Asn Phe Arg Leu Leu Gly
Asn Val Leu Val 100 105 110 Cys Val Leu Ala His His Phe Gly Lys Glu
Phe Thr Pro Pro Val Gln 115 120 125 Ala Ala Tyr Gln Lys Val Val Ala
Xaa Xaa Ala Asn Ala Leu Ala His 130 135 140 Lys Tyr Xaa 145
<210> SEQ ID NO 21 <211> LENGTH: 124 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 Gly
Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu 1 5 10
15 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Leu Glu Ser Phe Gly Asp
20 25 30 Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys
Ala His 35 40 45 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu
Ala His Leu Asp 50 55 60 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser
Glu Leu His Cys Asp Lys 65 70 75 80 Leu His Val Asp Pro Glu Asn Phe
Arg Leu Leu Gly Asn Val Leu Val 85 90 95 Cys Val Leu Ala His His
Phe Gly Lys Glu Phe Thr Pro Pro Val Gln 100 105 110 Ala Ala Tyr Gln
Lys Val Val Ala Gly Val Ala Asn 115 120 <210> SEQ ID NO 22
<211> LENGTH: 147 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 22 Met Val His Leu Thr Pro Glu
Glu Lys Ser Ala Val Thr Ala Leu Trp 1 5 10 15 Gly Lys Val Asn Val
Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu 20 25 30 Leu Val Val
Tyr Pro Trp Thr Gln Arg Phe Leu Glu Ser Phe Gly Asp 35 40 45 Leu
Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His 50 55
60 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp
65 70 75 80 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys
Asp Lys 85 90 95 Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly
Asn Val Leu Val 100 105 110 Cys Val Leu Ala His His Phe Gly Lys Glu
Phe Thr Pro Pro Val Gln 115 120 125 Ala Ala Tyr Gln Lys Val Val Ala
Gly Val Ala Asn Ala Leu Ala His 130 135 140 Lys Tyr His 145
<210> SEQ ID NO 23 <211> LENGTH: 80 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: N-terminal region of a consensus
sequence of mammalian transferrins <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (24)..(24) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (31)..(31) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (32)..(32) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (47)..(47) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (59)..(59) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (64)..(64) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Tyr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (65)..(65)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Leu <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (71)..(71) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (73)..(73) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (77)..(77) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (78)..(78) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <400> SEQUENCE: 23 Met Xaa Leu Ala Val Gly Ala
Leu Xaa Xaa Cys Ala Val Leu Xaa Leu 1 5 10 15 Cys Leu Ala Val Pro
Asp Lys Xaa Xaa Arg Trp Cys Ala Val Xaa Xaa 20 25 30 His Glu Ala
Thr Lys Cys Gln Ser Phe Xaa Asp His Met Lys Xaa Val 35 40 45 Ile
Pro Ser Asp Gly Pro Ser Val Ala Cys Xaa Lys Lys Ala Ser Xaa 50 55
60 Xaa Asp Cys Ile Arg Ala Xaa Ala Xaa Asn Glu Ala Xaa Xaa Val Thr
65 70 75 80 <210> SEQ ID NO 24 <211> LENGTH: 114
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: C-terminal
region of a consensus sequence of mammalian transferrins
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Thr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Asp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (18)..(18) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (23)..(23) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (28)..(28) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (30)..(30) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (61)..(61) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (62)..(62) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (75)..(75) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (78)..(78) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (96)..(96) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (97)..(97) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (105)..(105) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (107)..(107) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (110)..(110) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <400> SEQUENCE: 24 Glu Thr Xaa Glu Asp Cys Ile
Ala Lys Ile Met Asn Gly Glu Ala Xaa 1 5 10 15 Ala Xaa Xaa Leu Asp
Gly Xaa Phe Xaa Tyr Ile Xaa Gly Xaa Cys Gly 20 25 30 Leu Val Pro
Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp Asn Cys Glu 35 40 45 Asp
Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Xaa Xaa Lys Ser 50 55
60 Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Xaa Lys Lys Xaa Cys His
65 70 75 80 Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met Gly
Leu Xaa 85 90 95 Xaa Asn Lys Ile Asn His Cys Arg Xaa Asp Xaa Phe
Phe Xaa Glu Gly 100 105 110 Cys Ala <210> SEQ ID NO 25
<211> LENGTH: 698 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Consensus sequence of mammalian transferrins
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Arg <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid, preferably Val
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (15)..(15) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Gly <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (24)..(24)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Thr <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (25)..(25) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (31)..(31) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (32)..(32) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (47)..(47) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (59)..(59) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (64)..(64) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (71)..(71) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (73)..(73) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Ile <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (77)..(77)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Asp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (78)..(78) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (84)..(84) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (87)..(87) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (88)..(88) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (98)..(98) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (104)..(104) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (107)..(107) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (115)..(115) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (116)..(116) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (121)..(121) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (124)..(124) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (129)..(129) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (146)..(146) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (152)..(152) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (153)..(153) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (161)..(161) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (179)..(179) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (188)..(188) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (204)..(204) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (224)..(224) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (238)..(238) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (245)..(245) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (255)..(255) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (268)..(268) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably His <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (275)..(275) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (276)..(276) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (286)..(286) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (293)..(293) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (309)..(309) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (310)..(310) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (326)..(326) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (327)..(327) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (357)..(357) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (381)..(381) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (393)..(393) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (406)..(406) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (408)..(408) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (409)..(409) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (413)..(413) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (415)..(415) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (418)..(418) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (420)..(420) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (451)..(451) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (452)..(452) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (465)..(465) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (468)..(468) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (486)..(486) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (487)..(487) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (495)..(495) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (497)..(497) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (500)..(500) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (505)..(505) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (509)..(509) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (529)..(529) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (536)..(536) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (558)..(558) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (559)..(559) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (563)..(563) <223> OTHER INFORMATION: Xaa can be
any naturally occurring amino acid, preferably Gly <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(586)..(586) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Thr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(592)..(592) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Glu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(603)..(603) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Asn <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(607)..(607) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Val <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(622)..(622) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Gln <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(635)..(635) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Ser <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(648)..(648) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Leu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(652)..(652) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Asp <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(678)..(678) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Val <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(692)..(692) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Ala <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(696)..(696) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Arg <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(698)..(698) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Pro <400>
SEQUENCE: 25 Met Xaa Leu Ala Val Gly Ala Leu Xaa Xaa Cys Ala Val
Leu Xaa Leu 1 5 10 15 Cys Leu Ala Val Pro Asp Lys Xaa Xaa Arg Trp
Cys Ala Val Xaa Xaa 20 25 30 His Glu Ala Thr Lys Cys Gln Ser Phe
Xaa Asp His Met Lys Xaa Val 35 40 45 Ile Pro Ser Asp Gly Pro Ser
Val Ala Cys Xaa Lys Lys Ala Ser Xaa 50 55 60 Xaa Asp Cys Ile Arg
Ala Xaa Ala Xaa Asn Glu Ala Xaa Xaa Val Thr 65 70 75 80 Leu Asp Ala
Xaa Leu Val Xaa Xaa Ala Tyr Leu Ala Pro Xaa Asn Leu 85 90 95 Lys
Xaa Val Val Ala Glu Phe Xaa Gly Ser Xaa Xaa Asp Pro Gln Thr 100 105
110 Phe Tyr Xaa Xaa Val Ala Val Val Xaa Lys Asp Xaa Gly Phe Gln Met
115 120 125 Xaa Gln Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly
Arg Ser 130 135 140 Ala Xaa Trp Asn Ile Pro Ile Xaa Xaa Leu Tyr Cys
Asp Leu Pro Glu 145 150 155 160 Xaa Arg Lys Pro Leu Glu Lys Ala Val
Ala Asn Phe Phe Ser Gly Ser 165 170 175 Cys Ala Xaa Cys Ala Asp Gly
Thr Asp Phe Pro Xaa Leu Cys Gln Leu 180 185 190 Cys Pro Gly Cys Gly
Cys Ser Thr Leu Asn Gln Xaa Phe Gly Tyr Ser 195 200 205 Gly Ala Phe
Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Xaa 210 215 220 Lys
His Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Xaa Arg Asp 225 230
235 240 Gln Tyr Glu Leu Xaa Cys Leu Asp Asn Thr Arg Lys Pro Val Xaa
Glu 245 250 255 Tyr Lys Asp Cys His Leu Ala Gln Val Pro Ser Xaa Thr
Val Val Ala 260 265 270 Arg Ser Xaa Xaa Gly Lys Glu Asp Leu Ile Trp
Glu Leu Xaa Asn Gln 275 280 285 Ala Gln Glu His Xaa Gly Lys Asp Lys
Ser Lys Glu Phe Gln Leu Phe 290 295 300 Ser Ser Pro His Xaa Xaa Asp
Leu Leu Phe Lys Asp Ser Ala His Gly 305 310 315 320 Phe Leu Lys Val
Pro Xaa Xaa Met Asp Ala Lys Met Tyr Leu Gly Tyr 325 330 335 Glu Tyr
Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu 340 345 350
Ala Pro Thr Asp Xaa Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His 355
360 365 His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Xaa Val Gly
Lys 370 375 380 Ile Glu Cys Val Ser Ala Glu Thr Xaa Glu Asp Cys Ile
Ala Lys Ile 385 390 395 400 Met Asn Gly Glu Ala Xaa Ala Xaa Xaa Leu
Asp Gly Xaa Phe Xaa Tyr 405 410 415 Ile Xaa Gly Xaa Cys Gly Leu Val
Pro Val Leu Ala Glu Asn Tyr Asn 420 425 430 Lys Ser Asp Asn Cys Glu
Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val 435 440 445 Ala Val Xaa Xaa
Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys 450 455 460 Xaa Lys
Lys Xaa Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn 465 470 475
480 Ile Pro Met Gly Leu Xaa Xaa Asn Lys Ile Asn His Cys Arg Xaa Asp
485 490 495 Xaa Phe Phe Xaa Glu Gly Cys Ala Xaa Gly Ser Lys Xaa Asp
Ser Ser 500 505 510 Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn Leu
Cys Glu Pro Asn 515 520 525 Xaa Lys Glu Gly Tyr Tyr Gly Xaa Thr Gly
Ala Phe Arg Cys Leu Val 530 535 540 Glu Lys Gly Asp Val Ala Phe Val
Lys His Gln Thr Val Xaa Xaa Asn 545 550 555 560 Thr Gly Xaa Lys Asn
Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys 565 570 575 Asp Tyr Glu
Leu Leu Cys Leu Asp Gly Xaa Arg Lys Pro Val Glu Xaa 580 585 590 Tyr
Ala Asn Cys His Leu Ala Arg Ala Pro Xaa His Ala Val Xaa Thr 595 600
605 Arg Lys Asp Lys Glu Ala Cys Val His Lys Ile Leu Arg Xaa Gln Gln
610 615 620 His Leu Phe Gly Ser Asn Val Thr Asp Cys Xaa Gly Asn Phe
Cys Leu 625 630 635 640 Phe Arg Ser Glu Thr Lys Asp Xaa Leu Phe Arg
Xaa Asp Thr Val Cys 645 650 655 Leu Ala Lys Leu His Asp Arg Asn Thr
Tyr Glu Lys Tyr Leu Gly Glu 660 665 670 Glu Tyr Val Lys Ala Xaa Gly
Asn Leu Arg Lys Cys Ser Thr Ser Ser 675 680 685 Leu Leu Glu Xaa Cys
Thr Phe Xaa Arg Xaa 690 695 <210> SEQ ID NO 26 <211>
LENGTH: 80 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 26 Met Arg Leu Ala Val Gly Ala Leu Leu Val
Cys Ala Val Leu Gly Leu 1 5 10 15 Cys Leu Ala Val Pro Asp Lys Thr
Val Arg Trp Cys Ala Val Ser Glu 20 25 30 His Glu Ala Thr Lys Cys
Gln Ser Phe Arg Asp His Met Lys Ser Val 35 40 45 Ile Pro Ser Asp
Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr 50 55 60 Leu Asp
Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr 65 70 75 80
<210> SEQ ID NO 27 <211> LENGTH: 114 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Glu
Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly Glu Ala Asp 1 5 10
15 Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly Lys Cys Gly
20 25 30 Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp Asn
Cys Glu 35 40 45 Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val
Val Lys Lys Ser 50 55 60 Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys
Gly Lys Lys Ser Cys His 65 70 75 80 Thr Ala Val Gly Arg Thr Ala Gly
Trp Asn Ile Pro Met Gly Leu Leu 85 90 95 Tyr Asn Lys Ile Asn His
Cys Arg Phe Asp Glu Phe Phe Ser Glu Gly 100 105 110 Cys Ala
<210> SEQ ID NO 28 <211> LENGTH: 698 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 28 Met
Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu 1 5 10
15 Cys Leu Ala Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu
20 25 30 His Glu Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys
Ser Val 35 40 45 Ile Pro Ser Asp Gly Pro Ser Val Ala Cys Val Lys
Lys Ala Ser Tyr 50 55 60 Leu Asp Cys Ile Arg Ala Ile Ala Ala Asn
Glu Ala Asp Ala Val Thr 65 70 75 80 Leu Asp Ala Gly Leu Val Tyr Asp
Ala Tyr Leu Ala Pro Asn Asn Leu 85 90 95 Lys Pro Val Val Ala Glu
Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr 100 105 110 Phe Tyr Tyr Ala
Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met 115 120 125 Asn Gln
Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser 130 135 140
Ala Gly Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu 145
150 155 160 Pro Arg Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser
Gly Ser 165 170 175 Cys Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln
Leu Cys Gln Leu 180 185 190 Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn
Gln Tyr Phe Gly Tyr Ser 195 200 205 Gly Ala Phe Lys Cys Leu Lys Asp
Gly Ala Gly Asp Val Ala Phe Val 210 215 220 Lys His Ser Thr Ile Phe
Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp 225 230 235 240 Gln Tyr Glu
Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu 245 250 255 Tyr
Lys Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala 260 265
270 Arg Ser Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln
275 280 285 Ala Gln Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln
Leu Phe 290 295 300 Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys Asp
Ser Ala His Gly 305 310 315 320 Phe Leu Lys Val Pro Pro Arg Met Asp
Ala Lys Met Tyr Leu Gly Tyr 325 330 335 Glu Tyr Val Thr Ala Ile Arg
Asn Leu Arg Glu Gly Thr Cys Pro Glu 340 345 350 Ala Pro Thr Asp Glu
Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His 355 360 365 His Glu Arg
Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys 370 375 380 Ile
Glu Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile 385 390
395 400 Met Asn Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val
Tyr 405 410 415 Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu
Asn Tyr Asn 420 425 430 Lys Ser Asp Asn Cys Glu Asp Thr Pro Glu Ala
Gly Tyr Phe Ala Val 435 440 445 Ala Val Val Lys Lys Ser Ala Ser Asp
Leu Thr Trp Asp Asn Leu Lys 450 455 460 Gly Lys Lys Ser Cys His Thr
Ala Val Gly Arg Thr Ala Gly Trp Asn 465 470 475 480 Ile Pro Met Gly
Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp 485 490 495 Glu Phe
Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser 500 505 510
Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn 515
520 525 Asn Lys Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu
Val 530 535 540 Glu Lys Gly Asp Val Ala Phe Val Lys His Gln Thr Val
Pro Gln Asn 545 550 555 560 Thr Gly Gly Lys Asn Pro Asp Pro Trp Ala
Lys Asn Leu Asn Glu Lys 565 570 575 Asp Tyr Glu Leu Leu Cys Leu Asp
Gly Thr Arg Lys Pro Val Glu Glu 580 585 590 Tyr Ala Asn Cys His Leu
Ala Arg Ala Pro Asn His Ala Val Val Thr 595 600 605 Arg Lys Asp Lys
Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln 610 615 620 His Leu
Phe Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu 625 630 635
640 Phe Arg Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys
645 650 655 Leu Ala Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu
Gly Glu 660 665 670 Glu Tyr Val Lys Ala Val Gly Asn Leu Arg Lys Cys
Ser Thr Ser Ser 675 680 685 Leu Leu Glu Ala Cys Thr Phe Arg Arg Pro
690 695
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 28 <210>
SEQ ID NO 1 <211> LENGTH: 132 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Minimal consensus sequence of
mammalian ferritin H chain <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa at position 5 may be present or absent, if
present it means any naturally occurring amino acid, preferably Ile
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa at position 6
means any naturally occurring amino acid, preferably Asn
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: Xaa at position
14 means any naturally occurring amino acid, preferably Tyr
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION: Xaa at position
24 means any naturally occurring amino acid, preferably Tyr or Cys,
more preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (66)..(66) <223> OTHER
INFORMATION: Xaa at position 66 means any naturally occurring amino
acid, preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa at position 68 means any naturally occurring amino
acid, preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (75)..(75) <223> OTHER
INFORMATION: Xaa at position 75 means any naturally occurring amino
acid, preferably Arg or Cys, more preferably Cys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(90)..(90) <223> OTHER INFORMATION: Xaa at position 90 means
any naturally occurring amino acid, preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(94)..(94) <223> OTHER INFORMATION: Xaa at position 94 means
any naturally occurring amino acid, preferably Ser or Asn, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (120)..(120) <223> OTHER
INFORMATION: Xaa at position 120 may be present or absent, if
present it means any naturally occurring amino acid, preferably His
or Tyr, more preferably His <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (123)..(123)
<223> OTHER INFORMATION: Xaa at position 123 means any
naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (128)..(128) <223> OTHER
INFORMATION: Xaa at position 128 means any naturally occurring
amino acid, preferably Ala or Ser, more preferably Ala <400>
SEQUENCE: 1 Ser Glu Ala Ala Xaa Xaa Arg Gln Ile Asn Leu Glu Leu Xaa
Ala Ser 1 5 10 15 Tyr Val Tyr Leu Ser Met Ser Xaa Tyr Phe Asp Arg
Asp Asp Val Ala 20 25 30 Leu Lys Asn Phe Ala Lys Tyr Phe Leu His
Gln Ser His Glu Glu Arg 35 40 45 Glu His Ala Glu Lys Leu Met Lys
Leu Gln Asn Gln Arg Gly Gly Arg 50 55 60 Ile Xaa Leu Xaa Asp Ile
Lys Lys Pro Asp Xaa Asp Asp Trp Glu Ser 65 70 75 80 Gly Leu Asn Ala
Met Glu Cys Ala Leu Xaa Leu Glu Lys Xaa Asn Gln 85 90 95 Ser Leu
Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His 100 105 110
Leu Cys Asp Phe Ile Glu Thr Xaa Tyr Leu Xaa Glu Gln Val Lys Xaa 115
120 125 Ile Lys Glu Leu 130 <210> SEQ ID NO 2 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Consensus sequence of mammalian ferritin H chain <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(6)..(6) <223> OTHER INFORMATION: Xaa at position 6 can be
any naturally occurring amino acid, preferably Pro <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(14)..(14) <223> OTHER INFORMATION: Xaa at position 14 can be
any naturally occurring amino acid, preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(16)..(16) <223> OTHER INFORMATION: Xaa at position 16 can be
any naturally occurring amino acid, preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: Xaa at position 21 may be
present or absent, if present it means any naturally occurring
amino acid, preferably Ile <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (22)..(22) <223>
OTHER INFORMATION: Xaa at position 22 means any naturally occurring
amino acid, preferably Asn <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (30)..(30) <223>
OTHER INFORMATION: Xaa at position 30 can be any naturally
occurring amino acid, preferably Tyr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (40)..(40)
<223> OTHER INFORMATION: Xaa at position 40 can be any
naturally occurring amino acid, preferably Tyr or Cys, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (82)..(82) <223> OTHER
INFORMATION: Xaa at position 82 can be any naturally occurring
amino acid, preferably Phe <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (84)..(84) <223>
OTHER INFORMATION: Xaa at position 84 can be any naturally
occurring amino acid, preferably Gln <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (91)..(91)
<223> OTHER INFORMATION: Xaa at position 91 can be any
naturally occurring amino acid, preferably Arg or Cys, more
preferably Cys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (106)..(106) <223> OTHER
INFORMATION: Xaa at position 106 can be any naturally occurring
amino acid, preferably His <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (110)..(110)
<223> OTHER INFORMATION: Xaa at position 110 can be any
naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (137)..(137) <223> OTHER
INFORMATION: Xaa at position 137 can be any naturally occurring
amino acid, preferably His or Tyr, more preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(140)..(140) <223> OTHER INFORMATION: Xaa at position 140 can
be any naturally occurring amino acid, preferably Asn or Ser, more
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (145)..(145) <223> OTHER
INFORMATION: Xaa at position 145 can be any naturally occurring
amino acid, preferably Ala or Ser, more preferably Ala <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(164)..(164) <223> OTHER INFORMATION: Xaa at position 164 can
be any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (166)..(166) <223> OTHER
INFORMATION: Xaa at position 166 can be any naturally occurring
amino acid, preferably Met or Leu, prerferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(178)..(178) <223> OTHER INFORMATION: Xaa at position 178 can
be any naturally occurring amino acid, preferably Asp or His, more
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (181)..(181) <223> OTHER
INFORMATION: Xaa at position 181 is absent or any naturally
occurring amino acid, preferably Asn <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(182)..(182) <223> OTHER INFORMATION: Xaa at position 182 is
absent or any naturally occurring amino acid, preferably Glu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (183)..(183) <223> OTHER INFORMATION: Xaa at
position 183 is absent or any naturally occurring amino acid,
preferably Ser <400> SEQUENCE: 2 Met Thr Thr Ala Ser Xaa Ser
Gln Val Arg Gln Asn Tyr Xaa Gln Xaa 1 5 10 15 Ser Glu Ala Ala Xaa
Xaa Arg Gln Ile Asn Leu Glu Leu Xaa Ala Ser 20 25 30 Tyr Val Tyr
Leu Ser Met Ser Xaa Tyr Phe Asp Arg Asp Asp Val Ala 35 40 45 Leu
Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg 50 55
60 Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg
65 70 75 80 Ile Xaa Leu Xaa Asp Ile Lys Lys Pro Asp Xaa Asp Asp Trp
Glu Ser 85 90 95 Gly Leu Asn Ala Met Glu Cys Ala Leu Xaa Leu Glu
Lys Xaa Val Asn 100 105 110
Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro 115
120 125 His Leu Cys Asp Phe Ile Glu Thr Xaa Tyr Leu Xaa Glu Gln Val
Lys 130 135 140 Xaa Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg
Lys Met Gly 145 150 155 160 Ala Pro Glu Xaa Gly Xaa Ala Glu Tyr Leu
Phe Asp Lys His Thr Leu 165 170 175 Gly Xaa Ser Asp Xaa Xaa Xaa 180
<210> SEQ ID NO 3 <211> LENGTH: 133 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 3 Ala
Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser 1 5 10
15 Tyr Val Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp Val Ala
20 25 30 Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu
Glu Arg 35 40 45 Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln
Arg Gly Gly Arg 50 55 60 Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp
Arg Asp Asp Trp Glu Ser 65 70 75 80 Gly Leu Asn Ala Met Glu Cys Ala
Leu His Leu Glu Lys Ser Val Asn 85 90 95 Gln Ser Leu Leu Glu Leu
His Lys Leu Ala Thr Asp Lys Asn Asp Pro 100 105 110 His Leu Cys Asp
Phe Ile Glu Thr Tyr Tyr Leu Ser Glu Gln Val Lys 115 120 125 Ser Ile
Lys Glu Leu 130 <210> SEQ ID NO 4 <211> LENGTH: 180
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 4 Met Thr Thr Ala Ser Pro Ser Gln Val Arg Gln
Asn Tyr His Gln Asp 1 5 10 15 Ala Glu Ala Ala Ile Asn Arg Gln Ile
Asn Leu Glu Leu Tyr Ala Ser 20 25 30 Tyr Val Tyr Leu Ser Met Ser
Cys Tyr Phe Asp Arg Asp Asp Val Ala 35 40 45 Leu Lys Asn Phe Ala
Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg 50 55 60 Glu His Ala
Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg 65 70 75 80 Ile
Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp Asp Trp Glu Ser 85 90
95 Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Ser Val Asn
100 105 110 Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn
Asp Pro 115 120 125 His Leu Cys Asp Phe Ile Glu Thr Tyr Tyr Leu Ser
Glu Gln Val Lys 130 135 140 Ser Ile Lys Glu Leu Gly Asp His Val Thr
Asn Leu Arg Lys Met Gly 145 150 155 160 Ala Pro Glu Ala Gly Met Ala
Glu Tyr Leu Phe Asp Lys His Thr Leu 165 170 175 Gly His Gly Asp 180
<210> SEQ ID NO 5 <211> LENGTH: 132 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5 Ser
Glu Ala Ala Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr 1 5 10
15 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu
20 25 30 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu
Arg Glu 35 40 45 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg
Gly Gly Arg Ile 50 55 60 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys
Asp Asp Trp Glu Ser Gly 65 70 75 80 Leu Asn Ala Met Glu Cys Ala Leu
His Leu Glu Lys Asn Val Asn Gln 85 90 95 Ser Leu Leu Glu Leu His
Lys Leu Ala Thr Asp Lys Asn Asp Pro His 100 105 110 Leu Cys Asp Phe
Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala 115 120 125 Ile Lys
Glu Leu 130 <210> SEQ ID NO 6 <211> LENGTH: 179
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 Met Thr Thr Ala Ser Thr Ser Gln Val Arg Gln
Asn Tyr His Gln Asp 1 5 10 15 Ser Glu Ala Ala Asn Arg Gln Ile Asn
Leu Glu Leu Tyr Ala Ser Tyr 20 25 30 Val Tyr Leu Ser Met Ser Tyr
Tyr Phe Asp Arg Asp Asp Val Ala Leu 35 40 45 Lys Asn Phe Ala Lys
Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu 50 55 60 His Ala Glu
Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile 65 70 75 80 Phe
Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly 85 90
95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln
100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp
Pro His 115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu
Gln Val Lys Ala 130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn
Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu
Tyr Leu Phe Asp Lys His Thr Leu Gly 165 170 175 Asp Ser Asp
<210> SEQ ID NO 7 <211> LENGTH: 182 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Consensus sequence of mammalian
ferritin H chains 2nd version <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: Xaa at position 6 can be any naturally occurring
amino acid, preferably Pro <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (14)..(14) <223>
OTHER INFORMATION: Xaa at position 14 can be any naturally
occurring amino acid, preferably His <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Xaa at position 16 can be any
naturally occurring amino acid, preferably Asp <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Xaa at position 21 may be present or
absent, if present it means any naturally occurring amino acid,
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (29)..(29) <223> OTHER
INFORMATION: Xaa at position 29 can be any naturally occurring
amino acid, preferably Tyr <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (81)..(81) <223>
OTHER INFORMATION: Xaa at position 81 can be any naturally
occurring amino acid, preferably Phe <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (83)..(83)
<223> OTHER INFORMATION: Xaa at position 83 can be any
naturally occurring amino acid, preferably Gln <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(105)..(105) <223> OTHER INFORMATION: Xaa at position 105 can
be any naturally occurring amino acid, preferably His <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(144)..(144) <223> OTHER INFORMATION: Xaa at position 144 can
be any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (180)..(180) <223> OTHER
INFORMATION: Xaa at position 180 is absent or any naturally
occuring amino acid, preferably Asn <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(181)..(181) <223> OTHER INFORMATION: Xaa at position 181 is
absent or any naturally occuring amino acid, preferably Glu
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (182)..(182) <223> OTHER INFORMATION: Xaa at
position 182 is absent or any naturally occuring amino acid,
preferably Ser <400> SEQUENCE: 7 Met Thr Thr Ala Ser Xaa Ser
Gln Val Arg Gln Asn Tyr Xaa Gln Xaa 1 5 10 15
Ser Glu Ala Ala Xaa Arg Gln Ile Asn Leu Glu Leu Xaa Ala Ser Tyr 20
25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala
Leu 35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu
Glu Arg Glu 50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln
Arg Gly Gly Arg Ile 65 70 75 80 Xaa Leu Xaa Asp Ile Lys Lys Pro Asp
Cys Asp Asp Trp Glu Ser Gly 85 90 95 Leu Asn Ala Met Glu Cys Ala
Leu Xaa Leu Glu Lys Asn Val Asn Gln 100 105 110 Ser Leu Leu Glu Leu
His Lys Leu Ala Thr Asp Lys Asn Asp Pro His 115 120 125 Leu Cys Asp
Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Xaa 130 135 140 Ile
Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150
155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu
Gly 165 170 175 Asp Ser Asp Xaa Xaa Xaa 180 <210> SEQ ID NO 8
<211> LENGTH: 150 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Minimal consensus sequence of mammalian ferritin L
chain <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
at position 5 can be any naturally occurring amino acid, preferably
Asp or Glu, more preferably Asp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (12)..(12) <223>
OTHER INFORMATION: Xaa at position 12 can be any nnaturally
occurring amino acid, preferably Arg or Ser, more preferably Ser
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (17)..(17) <223> OTHER INFORMATION: Xaa at position
17 can be any naturally occurring amino acid, preferably Ser or
Arg, more preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa at position 19 can be any naturally occurring
amino acid, preferably Arg or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(29)..(29) <223> OTHER INFORMATION: Xaa at position 29 can be
any naturally occurring amino acid, preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(30)..(30) <223> OTHER INFORMATION: Xaa at position 30 can be
any naturally occurring amino acid, preferably Tyr or Phe, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: Xaa at position 42 can be any naturally occurring
amino acid, preferably Ser or Gly, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(56)..(56) <223> OTHER INFORMATION: Xaa at position 56 can be
any naturally occurirng amino acid, preferably Ala or Tyr, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (61)..(61) <223> OTHER
INFORMATION: Xaa at position 61 can be any naturally occurring
amino acid, preferably Glu or Lys, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(62)..(62) <223> OTHER INFORMATION: Xaa at position 62 can be
any naturally occurring amino acid, preferably Met or Phe, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa at position 65 can be any naturally occurring
amino acid, preferably Asp or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(75)..(75) <223> OTHER INFORMATION: Xaa at position 75 can be
any naturally occurring amino acid, preferably Ile or Val, more
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (76)..(76) <223> OTHER
INFORMATION: Xaa at position 76 can be any naturally occurring
amino acid, preferably Lys or Gln, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(79)..(79) <223> OTHER INFORMATION: Xaa at position 79 can be
any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (80)..(80) <223> OTHER
INFORMATION: Xaa at position 80 can be any naturally occurring
amino acid, preferably Glu or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(87)..(87) <223> OTHER INFORMATION: Xaa at position 87 can be
any naturally occurring amino acid, preferably Pro or Gln, more
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (88)..(88) <223> OTHER
INFORMATION: Xaa at position 88 can be any naturally occurring
amino acid, preferably Glu or Asp, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(91)..(91) <223> OTHER INFORMATION: Xaa at position 91 can be
any naturally occurring amino acid, preferably Glu or Lys, more
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa at position 94 can be any naturally occurring
amino acid, preferably Met or Leu, more preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(96)..(96) <223> OTHER INFORMATION: Xaa at position 96 can be
any naturally occurring amino acid, preferably Met or Leu, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (99)..(99) <223> OTHER
INFORMATION: Xaa at position 99 can be any naturally occurring
amino acid, preferably Lys or Asn, preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(115)..(115) <223> OTHER INFORMATION: Xaa at position 115 can
be any naturally occurring amino acid, preferably Thr or Ala, more
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (119)..(119) <223> OTHER
INFORMATION: Xaa at position 119 can be any naturally occurring
amino acid, preferably Leu <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (125)..(125)
<223> OTHER INFORMATION: Xaa at position 125 can be any
naturally occurring amino acid, preferably Ser or Thr, more
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (127)..(127) <223> OTHER
INFORMATION: Xaa at position 127 can be any naturally occurring
amino acid, preferably Tyr or Phe, more preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(130)..(130) <223> OTHER INFORMATION: Xaa at position 130 can
be any naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (140)..(140) <223> OTHER
INFORMATION: Xaa at position 140 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(146)..(146) <223> OTHER INFORMATION: Xaa at position 146 can
be any naturally occurring amino acid, preferably Arg or His, more
preferably His <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (148)..(148) <223> OTHER
INFORMATION: Xaa at position 148 can be any naturally occurring
amino acid, preferably Leu or Val, more preferably Leu <400>
SEQUENCE: 8 Asn Tyr Ser Thr Xaa Val Glu Ala Ala Val Asn Xaa Leu Val
Asn Leu 1 5 10 15 Xaa Leu Xaa Ala Ser Tyr Thr Tyr Leu Ser Leu Gly
Xaa Xaa Phe Asp 20 25 30 Arg Asp Asp Val Ala Leu Glu Gly Val Xaa
His Phe Phe Arg Glu Leu 35 40 45 Ala Glu Glu Lys Arg Glu Gly Xaa
Glu Arg Leu Leu Xaa Xaa Gln Asn 50 55 60 Xaa Arg Gly Gly Arg Ala
Leu Phe Gln Asp Xaa Xaa Lys Pro Xaa Xaa 65 70 75 80 Asp Glu Trp Gly
Lys Thr Xaa Xaa Ala Met Xaa Ala Ala Xaa Ala Xaa 85 90 95 Glu Lys
Xaa Leu Asn Gln Ala Leu Leu Asp Leu His Ala Leu Gly Ser 100 105 110
Ala Arg Xaa Asp Pro His Xaa Cys Asp Phe Leu Glu Xaa His Xaa Leu 115
120 125 Asp Xaa Glu Val Lys Leu Ile Lys Lys Met Gly Xaa His Leu Thr
Asn 130 135 140 Leu Xaa Arg Xaa Ala Gly 145 150 <210> SEQ ID
NO 9 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus sequence of mammalian ferritin L chain
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa at position
12 can be any naturally occurring amino acid, preferably Asp or
Glu, more preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa at position 19 can be any naturally occurring
amino acid, preferably Ser or Arg, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa at position 24 can be
any naturally occurring amino acid, preferably Ser or Arg, more
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa at position 26 can be any naturally occurring
amino acid, preferably Arg or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(36)..(36) <223> OTHER INFORMATION: Xaa at position 36 can be
any naturally
occurring amino acid, preferably Phe <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (37)..(37)
<223> OTHER INFORMATION: Xaa at position 37 can be any
naturally occurring amino acid, preferably Tyr or Phe, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (49)..(49) <223> OTHER
INFORMATION: Xaa at position 47 can be any naturally occurring
amino acid, preferably Ser or Gly, more preferably Ser <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(63)..(63) <223> OTHER INFORMATION: Xaa at position 63 can be
any naturally occurring amino acid, preferably Ala or Tyr, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa at position 68 can be any naturally occurring
amino acid, preferably Glu or Lys, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(69)..(69) <223> OTHER INFORMATION: Xaa at position 69 can be
any naturally occurring amino acid, preferably Met or Phe, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (72)..(72) <223> OTHER
INFORMATION: Xaa at position 72 can be any naturally occurring
amino acid, preferably Asp or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(82)..(82) <223> OTHER INFORMATION: Xaa at position 82 can be
any naturally occurring amino acid, preferably Ile or Val, more
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (83)..(83) <223> OTHER
INFORMATION: Xaa at position 83 can be any naturally occurring
amino acid, preferably Lys or Gln, more preferably Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(86)..(86) <223> OTHER INFORMATION: Xaa at position 86 can be
any naturally occurring amino acid, preferably Ala or Ser, more
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (87)..(87) <223> OTHER
INFORMATION: Xaa at position 87 can be any naturally occurring
amino acid, preferably Glu or Gln, more preferably Gln <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(94)..(94) <223> OTHER INFORMATION: Xaa at position 94 can be
any naturally occurring amino acid, preferably Pro or Gln, more
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (95)..(95) <223> OTHER
INFORMATION: Xaa at position 95 can be any naturally occurring
amino acid, preferably Glu or Asp, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(98)..(98) <223> OTHER INFORMATION: Xaa at position 98 can be
any naturally occurring amino acid, preferably Glu or Lys, more
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (101)..(101) <223> OTHER
INFORMATION: Xaa at position 101 can be any naturally occurring
amino acid, preferably Met or Leu, more preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(103)..(103) <223> OTHER INFORMATION: Xaa at position 103 can
be any naturally occurring amino acid, preferably Met or Leu, more
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (106)..(106) <223> OTHER
INFORMATION: Xaa at position 106 can be any naturally occurring
amino acid, preferably Lys or Asn, preferably Lys <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(122)..(122) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (126)..(126)
<223> OTHER INFORMATION: Xaa at position 126 can be any
naturally occurring amino acid, preferably Leu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(132)..(132) <223> OTHER INFORMATION: Xaa at position 132 can
be any naturally occurring amino acid, preferably Ser or Thr, more
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (134)..(134) <223> OTHER
INFORMATION: Xaa at position 134 can be any naturally occurring
amino acid, preferably Tyr or Phe, more preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(137)..(137) <223> OTHER INFORMATION: Xaa at position 137 can
be any naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (147)..(147) <223> OTHER
INFORMATION: Xaa at position 147 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(153)..(153) <223> OTHER INFORMATION: Xaa at position 153 can
be any naturally occurring amino acid, preferably Arg or His, more
preferably His <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (155)..(155) <223> OTHER
INFORMATION: Xaa at position 155 can be any naturally occurring
amino acid, preferably Leu or Val, more preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(161)..(161) <223> OTHER INFORMATION: Xaa at position 161 can
be absent or any naturally occurring amino acid, preferably Ala
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (163)..(163) <223> OTHER INFORMATION: Xaa at
position 163 can be absent or any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (166)..(166) <223> OTHER
INFORMATION: Xaa at position 166 can be absent or any naturally
occurring amino acid, preferably Pro <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(168)..(168) <223> OTHER INFORMATION: Xaa at position 168 can
be any naturally occurring amino acid, preferably Gly or Ala, more
preferably Ala <400> SEQUENCE: 9 Met Thr Ser Gln Ile Arg Gln
Asn Tyr Ser Thr Xaa Val Glu Ala Ala 1 5 10 15 Val Asn Xaa Leu Val
Asn Leu Xaa Leu Xaa Ala Ser Tyr Thr Tyr Leu 20 25 30 Ser Leu Gly
Xaa Xaa Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val 35 40 45 Xaa
His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Xaa Glu 50 55
60 Arg Leu Leu Xaa Xaa Gln Asn Xaa Arg Gly Gly Arg Ala Leu Phe Gln
65 70 75 80 Asp Xaa Xaa Lys Pro Xaa Xaa Asp Glu Trp Gly Lys Thr Xaa
Xaa Ala 85 90 95 Met Xaa Ala Ala Xaa Ala Xaa Glu Lys Xaa Leu Asn
Gln Ala Leu Leu 100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Xaa
Asp Pro His Xaa Cys Asp 115 120 125 Phe Leu Glu Xaa His Xaa Leu Asp
Xaa Glu Val Lys Leu Ile Lys Lys 130 135 140 Met Gly Xaa His Leu Thr
Asn Leu Xaa Arg Xaa Ala Gly Pro Gln Pro 145 150 155 160 Xaa Gln Xaa
Gly Val Xaa Gln Xaa Ser Leu Gly Glu Tyr Leu Phe Glu 165 170 175 Arg
Leu Thr Leu Lys His Asp 180 <210> SEQ ID NO 10 <211>
LENGTH: 150 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa
at position 5 can be any naturally occuring amino acid, preferably
Asp or Glu, more preferably Asp <400> SEQUENCE: 10 Asn Tyr
Ser Thr Glu Val Glu Ala Ala Val Asn Arg Leu Val Asn Leu 1 5 10 15
His Leu Arg Ala Ser Tyr Thr Tyr Leu Ser Leu Gly Phe Phe Phe Asp 20
25 30 Arg Asp Asp Val Ala Leu Glu Gly Val Gly His Phe Phe Arg Glu
Leu 35 40 45 Ala Glu Glu Lys Arg Glu Gly Ala Glu Arg Leu Leu Glu
Phe Gln Asn 50 55 60 Asp Arg Gly Gly Arg Ala Leu Phe Gln Asp Val
Gln Lys Pro Ser Gln 65 70 75 80 Asp Glu Trp Gly Lys Thr Gln Glu Ala
Met Glu Ala Ala Leu Ala Met 85 90 95 Glu Lys Asn Leu Asn Gln Ala
Leu Leu Asp Leu His Ala Leu Gly Ser 100 105 110 Ala Arg Ala Asp Pro
His Leu Cys Asp Phe Leu Glu Ser His Tyr Leu 115 120 125 Asp Lys Glu
Val Lys Leu Ile Lys Lys Met Gly Asn His Leu Thr Asn 130 135 140 Leu
Arg Arg Val Ala Gly 145 150 <210> SEQ ID NO 11 <400>
SEQUENCE: 11 000 <210> SEQ ID NO 12 <211> LENGTH: 149
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Asn Tyr Ser Thr Asp Val Glu Ala Ala Val
Asn Ser Leu Val Asn Leu 1 5 10 15 Tyr Leu Gln Ala Ser Tyr Thr Tyr
Leu Ser Leu Gly Phe Tyr Phe Asp 20 25 30 Arg Asp Asp Val Ala Leu
Glu Gly Val Ser His Phe Phe Arg Glu Leu 35 40 45 Ala Glu Glu Lys
Arg Glu Gly Tyr Glu Arg Leu Leu Lys Met Gln Asn 50 55 60
Gln Arg Gly Gly Arg Ala Leu Phe Gln Asp Ile Lys Lys Pro Ala Glu 65
70 75 80 Asp Glu Trp Gly Lys Thr Pro Asp Ala Met Lys Ala Ala Met
Ala Leu 85 90 95 Glu Lys Lys Leu Asn Gln Ala Leu Leu Asp Leu His
Ala Leu Gly Ser 100 105 110 Ala Arg Thr Asp Pro His Leu Cys Asp Phe
Leu Glu Thr His Phe Leu 115 120 125 Asp Glu Glu Val Lys Leu Ile Lys
Lys Met Gly Asp His Leu Thr Asn 130 135 140 Leu His Arg Leu Gly 145
<210> SEQ ID NO 13 <211> LENGTH: 175 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 Met
Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala 1 5 10
15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu
20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu
Gly Val 35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg
Glu Gly Tyr Glu 50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly
Gly Arg Ala Leu Phe Gln 65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp
Glu Trp Gly Lys Thr Pro Asp Ala 85 90 95 Met Lys Ala Ala Met Ala
Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu 100 105 110 Asp Leu His Ala
Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp 115 120 125 Phe Leu
Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys 130 135 140
Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145
150 155 160 Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu Thr Leu Lys His
Asp 165 170 175 <210> SEQ ID NO 14 <211> LENGTH: 183
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
seqeuence of mammaliam ferritin L chain 2nd version <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(36)..(36) <223> OTHER INFORMATION: Xaa at position 36 can be
any naturally occuring amino acid, preferably Phe <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(37)..(37) <223> OTHER INFORMATION: Xaa at position 37 can be
any naturally occuring amino acid, preferably Tyr or Phe, more
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa at position 94 can be any naturally occurring
amino acid, preferably Pro or Gln, more preferably Pro <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(126)..(126) <223> OTHER INFORMATION: Xaa at position 126 can
be any naturally occurring amino acid, preferably Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(137)..(137) <223> OTHER INFORMATION: Xaa at position 137 can
be any naturally occurring amino acid, preferably Lys or Glu, more
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (147)..(147) <223> OTHER
INFORMATION: Xaa at position 147 can be any naturally occurring
amino acid, preferably Asp or Asn, more preferably Asp <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(161)..(161) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (163)..(163)
<223> OTHER INFORMATION: Xaa at position 163 can be absent or
any naturally occurring amino acid, preferably Thr <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(166)..(166) <223> OTHER INFORMATION: Xaa at position 166 can
be absent or any naturally occurring amino acid, preferably Pro
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (168)..(168) <223> OTHER INFORMATION: Xaa at
position 168 can be any naturally occurring amino acid, preferably
Gly or Ala, more preferably Ala <400> SEQUENCE: 14 Met Thr
Ser Gln Ile Arg Gln Asn Tyr Ser Thr Glu Val Glu Ala Ala 1 5 10 15
Val Asn Arg Leu Val Asn Leu His Leu Arg Ala Ser Tyr Thr Tyr Leu 20
25 30 Ser Leu Gly Xaa Xaa Phe Asp Arg Asp Asp Val Ala Leu Glu Gly
Val 35 40 45 Gly His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu
Gly Ala Glu 50 55 60 Arg Leu Leu Lys Leu Gln Asn Gln Arg Gly Gly
Arg Ala Leu Phe Gln 65 70 75 80 Asp Val Gln Lys Pro Ala Gln Asp Glu
Trp Gly Lys Thr Xaa Glu Ala 85 90 95 Met Glu Ala Ala Leu Ala Leu
Glu Lys Asn Leu Asn Gln Ala Leu Leu 100 105 110 Asp Leu His Ala Leu
Gly Ser Ala Arg Thr Asp Pro His Xaa Cys Asp 115 120 125 Phe Leu Glu
Asn His Phe Leu Asp Xaa Glu Val Lys Leu Ile Lys Lys 130 135 140 Met
Gly Xaa His Leu Thr Asn Leu Arg Arg Leu Ala Gly Pro Gln Pro 145 150
155 160 Xaa Gln Xaa Gly Val Xaa Gln Xaa Ser Leu Gly Glu Tyr Leu Phe
Glu 165 170 175 Arg Leu Thr Leu Lys His Asp 180 <210> SEQ ID
NO 15 <211> LENGTH: 117 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Minimal consensus sequence of mammalian
haemoglobin alpha chain <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (41)..(41) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (48)..(48) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (49)..(49) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (55)..(55) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (59)..(59) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Aap <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (60)..(60) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (76)..(76) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE
<222> LOCATION: (79)..(79) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid, preferably Pro
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (90)..(90) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Leu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (91)..(91)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Val <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (94)..(94) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (102)..(102) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (117)..(117) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <400> SEQUENCE: 15 Val Gly Ala His Xaa Gly Glu
Tyr Xaa Ala Glu Ala Leu Glu Xaa Met 1 5 10 15 Phe Leu Ser Phe Xaa
Xaa Thr Lys Thr Tyr Phe Pro His Phe Asp Leu 20 25 30 Ser His Gly
Ser Ala Xaa Val Lys Xaa His Gly Lys Lys Val Ala Xaa 35 40 45 Xaa
Leu Thr Asn Ala Val Xaa His Val Asp Xaa Xaa Pro Asn Ala Leu 50 55
60 Ser Ala Leu Xaa Asp Leu His Ala His Lys Leu Xaa Val Asp Xaa Val
65 70 75 80 Asn Phe Lys Leu Leu Ser His Cys Leu Xaa Xaa Thr Leu Xaa
Ala His 85 90 95 Leu Pro Ala Glu Phe Xaa Pro Ala Val His Ala Xaa
Leu Asp Lys Phe 100 105 110 Leu Ala Ser Val Xaa 115 <210> SEQ
ID NO 16 <211> LENGTH: 142 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Consensus sequence of mammalian alpha chain
haemoglobins <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (17)..(17) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (32)..(32) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (38)..(38) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (39)..(39) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (55)..(55) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (58)..(58) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (66)..(66) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (72)..(72) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (76)..(76) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (77)..(77) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (85)..(85) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (93)..(93) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (96)..(96) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (107)..(107) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (111)..(111) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (119)..(119) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (125)..(125) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (134)..(134) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (142)..(142) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <400> SEQUENCE: 16 Xaa Xaa Leu Ser Pro Ala Asp
Lys Thr Asn Val Lys Ala Xaa Trp Gly 1 5 10 15 Xaa Val Gly Ala His
Xaa Gly Glu Tyr Xaa Ala Glu Ala Leu Glu Xaa 20 25 30 Met Phe Leu
Ser Phe Xaa Xaa Thr Lys Thr Tyr Phe Pro His Phe Asp 35 40 45 Leu
Ser His Gly Ser Ala Xaa Val Lys Xaa His Gly Lys Lys Val Ala 50 55
60 Xaa Xaa Leu Thr Asn Ala Val Xaa His Val Asp Xaa Xaa Pro Asn Ala
65 70 75 80 Leu Ser Ala Leu Xaa Asp Leu His Ala His Lys Leu Xaa Val
Asp Xaa
85 90 95 Val Asn Phe Lys Leu Leu Ser His Cys Leu Xaa Xaa Thr Leu
Xaa Ala 100 105 110 His Leu Pro Ala Glu Phe Xaa Pro Ala Val His Ala
Xaa Leu Asp Lys 115 120 125 Phe Leu Ala Ser Val Xaa Thr Val Leu Thr
Ser Lys Tyr Xaa 130 135 140 <210> SEQ ID NO 17 <211>
LENGTH: 117 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 17 Val Gly Ala His Ala Gly Glu Tyr
Gly Ala Glu Ala Leu Glu Arg Met 1 5 10 15 Phe Leu Ser Phe Pro Thr
Thr Lys Thr Tyr Phe Pro His Phe Asp Leu 20 25 30 Ser His Gly Ser
Ala Gln Val Lys Gly His Gly Lys Lys Val Ala Asp 35 40 45 Ala Leu
Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala Leu 50 55 60
Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro Val 65
70 75 80 Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala
Ala His 85 90 95 Leu Pro Ala Glu Phe Thr Pro Ala Val His Ala Ser
Leu Asp Lys Phe 100 105 110 Leu Ala Ser Val Ser 115 <210> SEQ
ID NO 18 <211> LENGTH: 142 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 Met Val Leu Ser Pro
Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly 1 5 10 15 Lys Val Gly
Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg 20 25 30 Met
Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp 35 40
45 Leu Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala
50 55 60 Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro
Asn Ala 65 70 75 80 Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu
Arg Val Asp Pro 85 90 95 Val Asn Phe Lys Leu Leu Ser His Cys Leu
Leu Val Thr Leu Ala Ala 100 105 110 His Leu Pro Ala Glu Phe Thr Pro
Ala Val His Ala Ser Leu Asp Lys 115 120 125 Phe Leu Ala Ser Val Ser
Thr Val Leu Thr Ser Lys Tyr Arg 130 135 140 <210> SEQ ID NO
19 <211> LENGTH: 124 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Minimal consensus sequence of mammalian
haemoglobin beta chain <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (14)..(14) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (27)..(27) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (35)..(35) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (36)..(36) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (49)..(49) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (52)..(52) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (66)..(66) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (69)..(69) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (86)..(86) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Glu <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (121)..(121) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (122)..(122) <223> OTHER INFORMATION: Xaa can be
absent or any naturally occurring amino acid, preferably Val
<400> SEQUENCE: 19 Gly Lys Val Xaa Val Asp Glu Val Xaa Xaa
Glu Xaa Xaa Xaa Xaa Leu 1 5 10 15 Leu Val Val Tyr Pro Trp Thr Gln
Arg Phe Xaa Glu Ser Phe Gly Asp 20 25 30 Leu Ser Xaa Xaa Asp Ala
Val Met Gly Asn Pro Lys Val Lys Ala His 35 40 45 Xaa Lys Lys Xaa
Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp 50 55 60 Asn Xaa
Lys Xaa Xaa Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys 65 70 75 80
Leu His Val Asp Pro Xaa Asn Phe Arg Leu Leu Gly Asn Val Leu Val 85
90 95 Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val
Gln 100 105 110 Ala Ala Tyr Gln Lys Val Val Ala Xaa Xaa Ala Asn 115
120 <210> SEQ ID NO 20 <211> LENGTH: 147 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Consensus sequence of
mammalian haemoglobin beta chain <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (11)..(11) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (20)..(20) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino
acid, preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (28)..(28) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (29)..(29) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (30)..(30) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (31)..(31) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (43)..(43) <223> OTHER
INFORMATION: Xaa can be absent or any naturally occurring amino
acid, preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (51)..(51) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (52)..(52) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (68)..(68) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (82)..(82) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (84)..(84) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (85)..(85) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (102)..(102) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (137)..(137) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (138)..(138) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (147)..(147) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably His <400> SEQUENCE: 20 Met Val His Leu Thr Pro Glu
Glu Lys Xaa Xaa Val Thr Ala Xaa Trp 1 5 10 15 Gly Lys Val Xaa Val
Asp Glu Val Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu 20 25 30 Leu Val Val
Tyr Pro Trp Thr Gln Arg Phe Xaa Glu Ser Phe Gly Asp 35 40 45 Leu
Ser Xaa Xaa Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His 50 55
60 Xaa Lys Lys Xaa Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp
65 70 75 80 Asn Xaa Lys Xaa Xaa Phe Ala Thr Leu Ser Glu Leu His Cys
Asp Lys 85 90 95 Leu His Val Asp Pro Xaa Asn Phe Arg Leu Leu Gly
Asn Val Leu Val 100 105 110 Cys Val Leu Ala His His Phe Gly Lys Glu
Phe Thr Pro Pro Val Gln 115 120 125 Ala Ala Tyr Gln Lys Val Val Ala
Xaa Xaa Ala Asn Ala Leu Ala His 130 135 140 Lys Tyr Xaa 145
<210> SEQ ID NO 21 <211> LENGTH: 124 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 Gly
Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu 1 5 10
15 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Leu Glu Ser Phe Gly Asp
20 25 30 Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys
Ala His 35 40 45 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu
Ala His Leu Asp 50 55 60 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser
Glu Leu His Cys Asp Lys 65 70 75 80 Leu His Val Asp Pro Glu Asn Phe
Arg Leu Leu Gly Asn Val Leu Val 85 90 95 Cys Val Leu Ala His His
Phe Gly Lys Glu Phe Thr Pro Pro Val Gln 100 105 110 Ala Ala Tyr Gln
Lys Val Val Ala Gly Val Ala Asn 115 120 <210> SEQ ID NO 22
<211> LENGTH: 147 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 22 Met Val His Leu Thr Pro Glu
Glu Lys Ser Ala Val Thr Ala Leu Trp 1 5 10 15 Gly Lys Val Asn Val
Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu 20 25 30 Leu Val Val
Tyr Pro Trp Thr Gln Arg Phe Leu Glu Ser Phe Gly Asp 35 40 45 Leu
Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His 50 55
60 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp
65 70 75 80 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys
Asp Lys 85 90 95 Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly
Asn Val Leu Val 100 105 110 Cys Val Leu Ala His His Phe Gly Lys Glu
Phe Thr Pro Pro Val Gln 115 120 125 Ala Ala Tyr Gln Lys Val Val Ala
Gly Val Ala Asn Ala Leu Ala His 130 135 140 Lys Tyr His 145
<210> SEQ ID NO 23 <211> LENGTH: 80 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: N-terminal region of a consensus
sequence of mammalian transferrins <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(9) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (24)..(24) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (31)..(31) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (32)..(32) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (47)..(47) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (59)..(59) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (64)..(64) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Tyr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (65)..(65)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Leu <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (71)..(71) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ile <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (73)..(73) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (77)..(77) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (78)..(78) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <400> SEQUENCE: 23 Met Xaa Leu Ala Val Gly Ala
Leu Xaa Xaa Cys Ala Val Leu Xaa Leu 1 5 10 15 Cys Leu Ala Val Pro
Asp Lys Xaa Xaa Arg Trp Cys Ala Val Xaa Xaa 20 25 30 His Glu Ala
Thr Lys Cys Gln Ser Phe Xaa Asp His Met Lys Xaa Val 35 40 45 Ile
Pro Ser Asp Gly Pro Ser Val Ala Cys Xaa Lys Lys Ala Ser Xaa 50 55
60 Xaa Asp Cys Ile Arg Ala Xaa Ala Xaa Asn Glu Ala Xaa Xaa Val Thr
65 70 75 80 <210> SEQ ID NO 24 <211> LENGTH: 114
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: C-terminal
region of a consensus sequence of mammalian transferrins
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (3)..(3) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Thr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (16)..(16)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Asp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (18)..(18) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (23)..(23) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (28)..(28) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (30)..(30) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (61)..(61) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (62)..(62) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (75)..(75) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (78)..(78) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (96)..(96) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (97)..(97) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (105)..(105) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (107)..(107) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (110)..(110) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <400> SEQUENCE: 24 Glu Thr Xaa Glu Asp Cys Ile
Ala Lys Ile Met Asn Gly Glu Ala Xaa 1 5 10 15 Ala Xaa Xaa Leu Asp
Gly Xaa Phe Xaa Tyr Ile Xaa Gly Xaa Cys Gly 20 25 30 Leu Val Pro
Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp Asn Cys Glu 35 40 45 Asp
Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Xaa Xaa Lys Ser 50 55
60 Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Xaa Lys Lys Xaa Cys His
65 70 75 80 Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met Gly
Leu Xaa 85 90 95 Xaa Asn Lys Ile Asn His Cys Arg Xaa Asp Xaa Phe
Phe Xaa Glu Gly 100 105 110 Cys Ala <210> SEQ ID NO 25
<211> LENGTH: 698 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Consensus sequence of mammalian transferrins
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Arg <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa
can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (15)..(15) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (24)..(24) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (25)..(25) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (31)..(31) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (32)..(32) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (42)..(42) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (47)..(47) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (59)..(59) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (64)..(64) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (71)..(71) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (73)..(73) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Ile <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (77)..(77)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid, preferably Asp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (78)..(78) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (84)..(84) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (87)..(87) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (88)..(88) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (94)..(94) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (98)..(98) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (104)..(104) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (107)..(107) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (108)..(108) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (115)..(115) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (116)..(116) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (121)..(121) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (124)..(124) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (129)..(129) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (146)..(146) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (152)..(152) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (153)..(153) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (161)..(161) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (179)..(179) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (188)..(188) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gln <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (204)..(204) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (224)..(224) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (238)..(238) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (245)..(245) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (255)..(255) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (268)..(268) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably His <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (275)..(275) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (276)..(276) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (286)..(286) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (293)..(293) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (309)..(309) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (310)..(310) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (326)..(326) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (327)..(327) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (357)..(357) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (381)..(381) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (393)..(393) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (406)..(406) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (408)..(408) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Met <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (409)..(409) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (413)..(413) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (415)..(415) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (418)..(418) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ala <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (420)..(420) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (451)..(451) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Val <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (452)..(452) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (465)..(465) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Gly <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (468)..(468) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (486)..(486) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Leu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (487)..(487) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (495)..(495) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (497)..(497) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Glu <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (500)..(500) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Ser <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (505)..(505) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (509)..(509) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (529)..(529) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Asn <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (536)..(536) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Tyr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (558)..(558) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (559)..(559) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (563)..(563) <223> OTHER INFORMATION: Xaa can be
any naturally occurring amino acid, preferably Gly <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(586)..(586) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Thr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(592)..(592) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Glu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(603)..(603) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Asn <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(607)..(607) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Val <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(622)..(622) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Gln <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(635)..(635) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Ser <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(648)..(648) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Leu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(652)..(652) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Asp <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(678)..(678) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Val <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(692)..(692) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid, preferably Ala <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(696)..(696) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid,
preferably Arg <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (698)..(698) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid,
preferably Pro <400> SEQUENCE: 25 Met Xaa Leu Ala Val Gly Ala
Leu Xaa Xaa Cys Ala Val Leu Xaa Leu 1 5 10 15 Cys Leu Ala Val Pro
Asp Lys Xaa Xaa Arg Trp Cys Ala Val Xaa Xaa 20 25 30 His Glu Ala
Thr Lys Cys Gln Ser Phe Xaa Asp His Met Lys Xaa Val 35 40 45 Ile
Pro Ser Asp Gly Pro Ser Val Ala Cys Xaa Lys Lys Ala Ser Xaa 50 55
60 Xaa Asp Cys Ile Arg Ala Xaa Ala Xaa Asn Glu Ala Xaa Xaa Val Thr
65 70 75 80 Leu Asp Ala Xaa Leu Val Xaa Xaa Ala Tyr Leu Ala Pro Xaa
Asn Leu 85 90 95 Lys Xaa Val Val Ala Glu Phe Xaa Gly Ser Xaa Xaa
Asp Pro Gln Thr 100 105 110 Phe Tyr Xaa Xaa Val Ala Val Val Xaa Lys
Asp Xaa Gly Phe Gln Met 115 120 125 Xaa Gln Leu Arg Gly Lys Lys Ser
Cys His Thr Gly Leu Gly Arg Ser 130 135 140 Ala Xaa Trp Asn Ile Pro
Ile Xaa Xaa Leu Tyr Cys Asp Leu Pro Glu 145 150 155 160 Xaa Arg Lys
Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser 165 170 175 Cys
Ala Xaa Cys Ala Asp Gly Thr Asp Phe Pro Xaa Leu Cys Gln Leu 180 185
190 Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn Gln Xaa Phe Gly Tyr Ser
195 200 205 Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala
Phe Xaa 210 215 220 Lys His Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys
Ala Xaa Arg Asp 225 230 235 240 Gln Tyr Glu Leu Xaa Cys Leu Asp Asn
Thr Arg Lys Pro Val Xaa Glu 245 250 255 Tyr Lys Asp Cys His Leu Ala
Gln Val Pro Ser Xaa Thr Val Val Ala 260 265 270 Arg Ser Xaa Xaa Gly
Lys Glu Asp Leu Ile Trp Glu Leu Xaa Asn Gln 275 280 285 Ala Gln Glu
His Xaa Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe 290 295 300 Ser
Ser Pro His Xaa Xaa Asp Leu Leu Phe Lys Asp Ser Ala His Gly 305 310
315 320 Phe Leu Lys Val Pro Xaa Xaa Met Asp Ala Lys Met Tyr Leu Gly
Tyr 325 330 335 Glu Tyr Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr
Cys Pro Glu 340 345 350 Ala Pro Thr Asp Xaa Cys Lys Pro Val Lys Trp
Cys Ala Leu Ser His 355 360 365 His Glu Arg Leu Lys Cys Asp Glu Trp
Ser Val Asn Xaa Val Gly Lys 370 375 380 Ile Glu Cys Val Ser Ala Glu
Thr Xaa Glu Asp Cys Ile Ala Lys Ile 385 390 395 400 Met Asn Gly Glu
Ala Xaa Ala Xaa Xaa Leu Asp Gly Xaa Phe Xaa Tyr 405 410 415 Ile Xaa
Gly Xaa Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn 420 425 430
Lys Ser Asp Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val 435
440 445 Ala Val Xaa Xaa Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu
Lys 450 455 460 Xaa Lys Lys Xaa Cys His Thr Ala Val Gly Arg Thr Ala
Gly Trp Asn 465 470 475 480 Ile Pro Met Gly Leu Xaa Xaa Asn Lys Ile
Asn His Cys Arg Xaa Asp 485 490 495 Xaa Phe Phe Xaa Glu Gly Cys Ala
Xaa Gly Ser Lys Xaa Asp Ser Ser 500 505 510 Leu Cys Lys Leu Cys Met
Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn 515 520 525 Xaa Lys Glu Gly
Tyr Tyr Gly Xaa Thr Gly Ala Phe Arg Cys Leu Val 530 535 540 Glu Lys
Gly Asp Val Ala Phe Val Lys His Gln Thr Val Xaa Xaa Asn 545 550 555
560 Thr Gly Xaa Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys
565 570 575 Asp Tyr Glu Leu Leu Cys Leu Asp Gly Xaa Arg Lys Pro Val
Glu Xaa 580 585 590 Tyr Ala Asn Cys His Leu Ala Arg Ala Pro Xaa His
Ala Val Xaa Thr 595 600 605 Arg Lys Asp Lys Glu Ala Cys Val His Lys
Ile Leu Arg Xaa Gln Gln 610 615 620 His Leu Phe Gly Ser Asn Val Thr
Asp Cys Xaa Gly Asn Phe Cys Leu 625 630 635 640 Phe Arg Ser Glu Thr
Lys Asp Xaa Leu Phe Arg Xaa Asp Thr Val Cys 645 650 655 Leu Ala Lys
Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu 660 665 670 Glu
Tyr Val Lys Ala Xaa Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser 675 680
685 Leu Leu Glu Xaa Cys Thr Phe Xaa Arg Xaa 690 695 <210> SEQ
ID NO 26 <211> LENGTH: 80 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 26 Met Arg Leu Ala Val
Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu 1 5 10 15 Cys Leu Ala
Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu 20 25 30 His
Glu Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val 35 40
45 Ile Pro Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr
50 55 60 Leu Asp Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala
Val Thr 65 70 75 80 <210> SEQ ID NO 27 <211> LENGTH:
114 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 27 Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile
Met Asn Gly Glu Ala Asp 1 5 10 15 Ala Met Ser Leu Asp Gly Gly Phe
Val Tyr Ile Ala Gly Lys Cys Gly 20 25 30 Leu Val Pro Val Leu Ala
Glu Asn Tyr Asn Lys Ser Asp Asn Cys Glu 35 40 45 Asp Thr Pro Glu
Ala Gly Tyr Phe Ala Val Ala Val Val Lys Lys Ser 50 55 60 Ala Ser
Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys Ser Cys His 65 70 75 80
Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu 85
90 95 Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe Ser Glu
Gly 100 105 110 Cys Ala <210> SEQ ID NO 28 <211>
LENGTH: 698 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 28 Met Arg Leu Ala Val Gly Ala Leu
Leu Val Cys Ala Val Leu Gly Leu 1 5 10 15 Cys Leu Ala Val Pro Asp
Lys Thr Val Arg Trp Cys Ala Val Ser Glu 20 25 30 His Glu Ala Thr
Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val 35 40 45 Ile Pro
Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr 50 55 60
Leu Asp Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr 65
70 75 80 Leu Asp Ala Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn
Asn Leu 85 90 95 Lys Pro Val Val Ala Glu Phe Tyr Gly Ser Lys Glu
Asp Pro Gln Thr 100 105 110 Phe Tyr Tyr Ala Val Ala Val Val Lys Lys
Asp Ser Gly Phe Gln Met 115 120 125 Asn Gln Leu Arg Gly Lys Lys Ser
Cys His Thr Gly Leu Gly Arg Ser 130 135 140 Ala Gly Trp Asn Ile Pro
Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu 145 150 155 160 Pro Arg Lys
Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser 165 170 175 Cys
Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu 180 185
190 Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser
195 200 205 Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala
Phe Val 210 215 220 Lys His Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys
Ala Asp Arg Asp 225 230 235 240 Gln Tyr Glu Leu Leu Cys Leu Asp Asn
Thr Arg Lys Pro Val Asp Glu 245 250 255
Tyr Lys Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala 260
265 270 Arg Ser Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn
Gln 275 280 285 Ala Gln Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe
Gln Leu Phe 290 295 300 Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys
Asp Ser Ala His Gly 305 310 315 320 Phe Leu Lys Val Pro Pro Arg Met
Asp Ala Lys Met Tyr Leu Gly Tyr 325 330 335 Glu Tyr Val Thr Ala Ile
Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu 340 345 350 Ala Pro Thr Asp
Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His 355 360 365 His Glu
Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys 370 375 380
Ile Glu Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile 385
390 395 400 Met Asn Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe
Val Tyr 405 410 415 Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala
Glu Asn Tyr Asn 420 425 430 Lys Ser Asp Asn Cys Glu Asp Thr Pro Glu
Ala Gly Tyr Phe Ala Val 435 440 445 Ala Val Val Lys Lys Ser Ala Ser
Asp Leu Thr Trp Asp Asn Leu Lys 450 455 460 Gly Lys Lys Ser Cys His
Thr Ala Val Gly Arg Thr Ala Gly Trp Asn 465 470 475 480 Ile Pro Met
Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp 485 490 495 Glu
Phe Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser 500 505
510 Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn
515 520 525 Asn Lys Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys
Leu Val 530 535 540 Glu Lys Gly Asp Val Ala Phe Val Lys His Gln Thr
Val Pro Gln Asn 545 550 555 560 Thr Gly Gly Lys Asn Pro Asp Pro Trp
Ala Lys Asn Leu Asn Glu Lys 565 570 575 Asp Tyr Glu Leu Leu Cys Leu
Asp Gly Thr Arg Lys Pro Val Glu Glu 580 585 590 Tyr Ala Asn Cys His
Leu Ala Arg Ala Pro Asn His Ala Val Val Thr 595 600 605 Arg Lys Asp
Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln 610 615 620 His
Leu Phe Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu 625 630
635 640 Phe Arg Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val
Cys 645 650 655 Leu Ala Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr
Leu Gly Glu 660 665 670 Glu Tyr Val Lys Ala Val Gly Asn Leu Arg Lys
Cys Ser Thr Ser Ser 675 680 685 Leu Leu Glu Ala Cys Thr Phe Arg Arg
Pro 690 695
* * * * *
References