U.S. patent application number 11/401005 was filed with the patent office on 2006-10-12 for il-21 derivatives and variants.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Florencio Zaragoza Dorwald, Bernd Peschke, Christine Bruun Schiodt, Helle Woldike, Anne Worsaae.
Application Number | 20060228331 11/401005 |
Document ID | / |
Family ID | 34437790 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060228331 |
Kind Code |
A1 |
Peschke; Bernd ; et
al. |
October 12, 2006 |
IL-21 Derivatives and variants
Abstract
The invention provides derivatives of IL-21 or variants thereof,
methods of producing such variants, new variants of IL-21, and
various methods of using such molecules.
Inventors: |
Peschke; Bernd; (Malov,
DK) ; Schiodt; Christine Bruun; (Bronshoj, DK)
; Woldike; Helle; (Lynge, DK) ; Dorwald; Florencio
Zaragoza; (Smorum, DK) ; Worsaae; Anne;
(Lyngby, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;PATENT DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
34437790 |
Appl. No.: |
11/401005 |
Filed: |
April 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/DK04/00686 |
Oct 8, 2004 |
|
|
|
11401005 |
Apr 10, 2006 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
435/320.1; 435/325; 435/69.52; 530/351; 536/23.5 |
Current CPC
Class: |
A61K 38/20 20130101;
A61K 47/60 20170801; C07K 14/54 20130101; A61P 35/00 20180101; A61P
35/02 20180101 |
Class at
Publication: |
424/085.2 ;
435/069.52; 435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C07H 21/04 20060101 C07H021/04; C12P 21/04 20060101
C12P021/04; C07K 14/54 20060101 C07K014/54 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2003 |
DK |
PA 2003 01496 |
Oct 17, 2003 |
DK |
PA 2003 01529 |
May 4, 2004 |
DK |
PA 2004 00707 |
Claims
1. A derivative of interleukin 21 (IL-21) or an IL-21 variant
comprising at least one polymeric molecule or lipophilic
substituent.
2. The derivative of claim 1, wherein the polymeric molecule is one
or more PEG groups.
3. The derivative of claim 2, wherein the polymeric molecule is one
or more PEG groups having a weight of at least about 5 kDa.
4. The derivative of claim 3, wherein the derivative comprises one
or more PEG groups having a weight of at least about 30 kDa.
5. The derivative of claim 1, wherein the polymeric molecule or
lipophilic derivative is conjugated to the amino acid sequence of
the IL-21 or IL-21 variant at the C-terminus.
6. The derivative of claim 1, wherein the polymeric molecule or
lipophilic derivative is conjugated to the interior of the amino
acid sequence of the IL-21 or IL-21 variant.
7. The derivative of claim 1, wherein the polymeric molecule or
lipophilic derivative is conjugated to the amino acid sequence of
the IL-21 or IL-21 variant at the N-terminus.
8. The derivative of claim 1, wherein the IL-21 or IL-21 variant is
an IL-21.
9. The derivative of claim 1, wherein the IL-21 or IL-21 variant is
an IL-21 variant.
10. The derivative of claim 9, wherein the polymeric molecule or
lipophilic derivative is conjugated to the IL-21 variant at an
amino acid that does not naturally occur in human IL-21.
11. A variant of interleukin 21 (IL-21) comprising an N-terminal
addition of a sequence of 1-10 amino acids comprising a Ser, Tyr,
Lys, or Cys residue.
12. A variant of human interleukin 21 (hIL-21) comprising an
addition of a serine residue at the N-terminus of the human IL-21
amino acid sequence (Ser-hIL-21).
13. The IL-21 variant of claim 12, wherein the IL-21 variant is
derivatized by conjugation of a polymeric group or lipophilic
substituent to the N-terminal Ser residue of hIL-21.
14. An isolated DNA comprising a sequence that codes for the
expression of Ser-hIL-21.
15. A method of producing an IL-21 variant derivative comprising
derivatizing Ser-hIL-21 with a polymeric molecule under conditions
suitable for producing a derivative of Ser-hIL-21.
16. A method of treating cancer comprising administering to a
patient in need thereof an effective amount of a derivative
according to claim 13.
17. A method of treating infection comprising administering to a
patient in need thereof an effective amount of a derivative
according to claim 13.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This patent application is a continuation of copending
International Patent Application No. PCT/DK2004/000686 (published
as WO 2005/035565), filed Oct. 8, 2004 (which designates the US)
and claims the benefit (under 35 USC .sctn.119) of U.S. Provisional
Patent Applications 60/510,892, 60/513,422, and 60/569,566, filed
Oct. 14, 2003, Oct. 22, 2003, and May 10, 2004, respectively, and
Danish Patent Applications PA 2003 01496, PA 2003 01529, and PA
2004 00707, filed Oct. 10, 2003, Oct. 17, 2003, and May 4, 2004,
respectively, the entirely of each of which is hereby incorporated
by reference.
FIELD OF THE INVENTION
[0002] The invention relates to derivatives of interleukin 21
(IL-21) and IL-21 variants, as well as the synthesis and
purification of derivatives of IL-21 and analogues thereof
(including variants of IL-21 which act as IL-21 antagonists).
BACKGROUND OF THE INVENTION
[0003] IL-21 was described for example in WO 00/53761 as a
stimulator of T cell growth and NK activity. Derivatives of IL-21
have previously not been described. As a potent pharmaceutical
derivatives with modified characteristics are interesting in many
applications.
SUMMARY OF THE INVENTION
[0004] The invention provides derivatives of IL-21 or variants
thereof.
[0005] In an aspect the invention provides derivatives of IL-21 or
variants thereof which comprises a polymeric molecule or lipophilic
derivative (substituent).
[0006] In an aspect of the invention the derivative of IL-21 or
variants thereof, comprises a polymeric molecule which is one or
more PEG groups.
[0007] In an aspect of the invention derivatives of IL-21 or
variants thereof, comprises derivatisation in the N-terminal, or
the C-terminal or internally in the molecule.
[0008] In an aspect of the invention the derivatisation is on a
naturally occuring amino acid, and/or also or alternatively one an
aminoacid added or substituted into the IL-21 sequence.
[0009] The invention provides the specific variant Ser-hIL21,
isolated DNA expressing the specific variant and the use for
derivatisation with a polymeric molecule. The invention also
provides the use of the derivatives of IL-21 or variants thereof,
for the manufacture of a medicament for the treatment of cancer of
infectious diseases.
DESCRIPTION OF THE INVENTION
[0010] The invention provides various derivatives of the IL-21
peptides. The derivatives include chemically modified peptides that
comprise an IL-21 peptide, or variants of the IL-21 peptide.
Chemical modification may alter the chemical and biological
characteristics of a molecule dependent on the characteristics of
the derivatising molecule. The effect of modification may be
maintaining the biological function of the peptide or potentially a
lower activity of the peptide. For example derivatisation may
extend the functional in vivo half life of a derivatised peptide
and thus compensate for a lower activity. For example a protracted
profile effect of IL-21 derivatives may be achieved by coupling of
a IL-21 peptide or an analogue thereof to a hydrophilic moiety that
results in IL-21 derivatives with a maintained biological activity.
The derivatisation may for example provide a peptide with an
improved half-life, thereby facilitating the continuous presence of
therapeutically effective amount of IL-21 or a derivative thereof
having the same biological effect. The amount needed for
administration of an effective amount of a protracted peptide may
thus be lower. Derivatisation may protect the molecule against
degradation by enzymes and prevent clearence from the body. The
derivatisation is preferably non-immugenic. In an aspect of the
invention the solubility of the peptide may be amended.
[0011] IL-21 activity is as defined as described in Parrish-Novak,
Nature, 408, 57-63, 2000; Brady, J., Hayakawa, Y., Smyth, M. J.,
and Nutt, S. L. 2004. IL-21 induces the functional maturation of
murine NK cells. Journal of immunology (Baltimore, Md.
172:2048-2058; Collins, M., Whitters, M. J., and Young, D. A. 2003.
IL-21 and IL-21 receptor: a new cytokine pathway modulates innate
and adaptive immunity. Immunol Res 28:131-140; Habib, T., Nelson,
A., and Kaushansky, K. 2003. IL-21: a novel IL-2-family lymphokine
that modulates B, T, and natural killer cell responses. J Allergy
Clin Immunol 112:1033-1045. Sivakumar, P. V. 2004. Interleukin-21
is a T-helper cytokine that regulates humoral immunity and
cell-mediated anti-tumour responses. Immunology 112:177; Wang, G.,
Tschoi, M., Spolski, R., Lou, Y., Ozaki, K., Feng, C., Kim, G.,
Leonard, W. J., and Hwu, P. 2003. In vivo antitumor activity of
interleukin 21 mediated by natural killer cells. Cancer Res
63:9016-9022; Wang, G. 2003. In vivo antitumor activity of
interleukin 21 mediated by natural killer cells. Cancer Res
63:9016. IL-21 and derivatives thereof are considered useful in the
treatment of neoplastic disorders. Neoplastic disorders or cancer
are to be understood as referring to all forms of neoplastic cell
growth, including both cystic and solid tumors, bone and soft
tissue tumors, including both benign and malignant tumors,
including tumors in anal tissue, bile duct, bladder, blood cells,
bone, bone (secondary), bowel (colon & rectum), brain, brain
(secondary), breast, breast (secondary), carcinoid, cervix,
children's cancers, eye, gullet (oesophagus), head & neck,
kaposi's sarcoma, kidney, larynx, leukaemia (acute lymphoblastic),
leukaemia (acute myeloid), leukaemia (chronic lymphocytic),
leukaemia (chronic myeloid), leukaemia (other), liver, liver
(secondary), lung, lung (secondary), lymph nodes (secondary),
lymphoma (hodgkin's), lymphoma (non-hodgkin's), melanoma,
mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin, soft
tissue sarcomas, stomach, testes, thyroid, unknown primary tumor,
vagina, vulva, womb (uterus). Soft tissue tumors include Benign
schwannoma Monosomy, Desmoid tumor, Lipo-blastoma, Lipoma, Uterine
leiomyoma, Clear cell sarcoma, Dermatofibrosarcoma, Ewing sarcoma,
Extraskeletal myxoid chondrosarcoma, Liposarcoma myxoid,
Liposarcoma, well differentiated, Alveolar rhabdomyosarcoma, and
Synovial sarcoma. Specific bone tumor include Nonossifying Fibroma,
Unicameral bone cyst, Enchon-droma, Aneurysmal bone cyst,
Osteoblastoma, Chondroblastoma, Chondromyxofibroma, Ossifying
fibroma and Adamantinoma, Giant cell tumor, Fibrous dysplasia,
Ewing's Sarcoma, Eosinophilic Granuloma, Osteosarcoma, Chondroma,
Chondrosarcoma, Malignant Fibrous Histiocytoma, and Metastatic
Carcinoma. Leukaemias referes to cancers of the white blood cells
which are produced by the bone marrow. This includes but are not
limited to the four main types of leukaemia; acute lymphoblastic
(ALL), acute myeloblastic (AML), chronic lymphocytic (CLL) and
chronic myeloid (CML).
[0012] Prior to a discussion of the detailed embodiments of the
invention, a definition of specific terms related to the main
aspects of the invention is provided.
[0013] In the context of the present invention IL-21 is defined as
the sequence disclosed in WO00/53761 as SEQ ID No.:2. or the same
sequence without the N-terminal sequence. The present application
also describes variants and derivatives of IL-21. In the context of
the present invention the term "IL-21" thus means IL-21as described
in WO00/53761 optionally without the N-terminal sequence. The
present invention embraces counterpart proteins and from other
species ("orthologs"). Of particular interest are IL-21
polypeptides from other mammalian species, including rodent,
porcine, ovine, bovine, canine, feline, equine, and other
primates.
[0014] "IL-21 derivatives" comprises derivatisation or linking to
another functional molecule. The linking can be chemical coupling,
genetic fusion, non-covalent association or the like, to other
molecular entities such as antibodies, toxins, radioisotope,
cytotoxic or cytostatic agents or polymeric molecules or lipophilic
groups. Non-limiting examples include polymeric groups such as,
e.g, dendrimers as disclosed in PCT/DK2004/000531, polyalkylene
oxide (PAO), polyalkylene glycol (PAG), polyethylene glycol (PEG),
polypropylene glycol (PPG), branched PEGs, polyvinyl alcohol (PVA),
polycarboxylate, poly-vinylpyrolidone, polyethylene-co-maleic acid
anhydride, polystyrene-co-maleic acid anhydride, dextran,
carboxymethyl-dextran; serum protein binding-ligands, such as
compounds which bind to albumin, like fatty acids, C.sub.5-C.sub.24
fatty acid, aliphatic diacid (e.g. C.sub.5-C.sub.24). Albumin
binders are described in Danish patent applications
PCT/DK04/000625. Albumin binders are also compounds of the
following formula: ##STR1## Other examples of protracting groups
includes small organic molecules containing moieties that under
physiological conditions alters charge properties, such as
carboxylic acids or amines, or neutral substituents that prevent
glycan specific recognition such as smaller alkyl substituents
(e.g., C.sub.1-C.sub.5 alkyl).
[0015] Variants or analogues of IL-21 peptides are characterized as
having one or more amino acid substitutions, deletions or
additions. These changes are typically of a minor nature, that is
conservative amino acid substitutions and other substitutions that
do not significantly affect the receptor binding, receptor
affinity, folding or biological activity of the peptide; However,
as described below even small amendments in essential aminoacids
changes the effect of the IL-21 peptide. Small deletions, typically
of one to about 30 amino acids; and small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25 residues, or a
small extension that facilitates purification (an affinity tag),
such as a poly-histidine tract, protein A, Nilsson et al., EMBO J.
4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991),
glutathione S transferase, Smith and Johnson, Gene 67:31 (1988), or
other antigenic epitope or binding domain. See, in general Ford et
al., Protein Expression and Purification 2: 95-107 (1991). DNAs
encoding affinity tags are available from commercial suppliers
(e.g., Pharmacia Biotech, Piscataway, N.J.). Variants of IL-21
peptides may also comprise non-naturally occurring amino acid
residues. Non-naturally occurring amino acids include, without
limitation, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans-4-hydroxyproline, Nmethylglycine,
addo-threonine, methylthreonine, hydroxyethylcysteine,
hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic
acid, thiazolidine carboxylic acid, dehydroproline, 3- and
4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and
4-fluorophenylalanine. Several methods are known in the art for
incorporating nonnaturally occurring amino acid residues into
proteins. For example, an in vitro system can be employed wherein
nonsense mutations are suppressed using chemically aminoacylated
suppressor tRNAs. Methods for synthesizing amino acids and
aminoacylating tRNA are known in the art. Essential amino acids in
the polypeptides of the present invention can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alaninescanning mutagenesis [Cunningham and Wells,
Science 244: 1081-1085 (1989)]; Bass et al., Proc. Natl. Acad. Sci.
USA 88:4498-4502 (1991). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity
(e.g., ligand binding and signal transduction) to identify amino
acid residues that are critical to the activity of the molecule.
Sites of ligand-protein interaction can also be determined by
analysis of crystal structure as determined by such techniques as
nuclear magnetic resonance, crystallography or photoaffinity
labeling. The identities of essential amino acids can also be
inferred from analysis of homologies with related proteins.
[0016] In one embodiment a variant is 70% or more identical with
the sequence of SEQ ID NO:2 of WO0053761. In one embodiment a
variant is 80% or more identical with the SEQ ID NO:2 of WO0053761.
In another embodiment a variant is 90% or more identical with the
sequence of SEQ ID NO:2 of WO0053761. In a further embodiment a
variant is 95% or more identical with the sequence of SEQ ID NO:2
of WO0053761.
[0017] Percentage sequence identity between two amino acid
sequences is determined by a Needelman-Wunsch alignment, useful for
both protein and DNA alignments. For protein alignments the default
scoring matrix used is BLOSUM50, and the penalty for the first
residue in a gap is -12, while the penalty for additional residues
in a gap is -2. The alignment may be made with the Align software
from the FASTA package version v20u6 (W. R. Pearson and D. J.
Lipman (1988), "Improved Tools for Biological Sequence Analysis",
PNAS 85:2444-2448; and W. R. Pearson (1990) "Rapid and Sensitive
Sequence Comparison with FASTP and FASTA", Methods in Enzymology,
183:63-98).
[0018] Non-limiting examples of IL-21 variants having substantially
modified biological activity relative to wild-type IL-21 is
described in WO03/40313 wherein substitutions of single amino acids
in the IL-21 sequence antagonises the effect of IL-21.
[0019] According to this invention antagonists of IL-21 compounds
which inhibit the activity normally observed with IL-21. Such
compounds may be as well small molecules, peptides or soluble
receptors interacting with IL-21. According to the present
invention derivatisation of the antagonists of IL-21 are peptides
or soluble receptors. In an aspect of the invention the peptides
are IL-21 analogues or variants having an antagonistic effect.
[0020] Antagonists are also fusion protein that includes the
extracellular domain of the IL-21 R fused to an Fc immunoglobulin
region, Examples of antagonistic fusion proteins are shown in SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ II) NO:31,
SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, of
WO03/28630.
[0021] Other IL-21 antagonists to be used according to the
invention are the sequences 4 and 6 of WO03/40313. Tthe IL-21
peptides with variations in one or both of the positions 114 and
119 as mentioned in WO03/87320.
[0022] Soluble receptors of IL-21 having antagonistic effect on
IL-21 are disclosed in WO04/07682.
[0023] Examples of antagonistic fusion proteins that can be used in
the methods of the invention are shown in SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, and SEQ ID NO:39, of WO03/28630.
[0024] Other IL-21 antagonists that can be used in inventive
methods provided here are SEQ ID NOS 4 and 6 of WO03/40313 and
IL-21 peptides with variations in one or both of the positions 114
and 119 of human IL-21 as mentioned in WO03/87320. WO04/07682
describes soluble receptors having antagonistic activity against
IL-21.
[0025] In an aspect of the invention, IL-21 antibodies are used as
IL-21 antagonists. Such antibodies can be produced by any suitable
method known in the art and examples of such antibodies are
described in WO00/53761. IL-21 antagonist antibodies are
characterised by inhibiting one or more biological activities of
IL-21. Inhibition of biologic activity can be measured by, e.g.,
the Ba F3 assay where Ba F3 cells stably transfected with human
IL-21 R (IL-21 R-Ba F3) undergo proliferation when IL-21 is added
to the culture. Addition of IL-21 antagonists to the IL-21 R-Ba F3
cells desirably partially or fully inhibits IL-21-dependent
proliferation of IL-21 R-Ba F3 cells.
[0026] The term "polymeric molecule", or "polymeric group" or
"polymeric moiety" or "polymer molecule", encompasses molecules
formed by covalent linkage of two or more monomers wherein none of
the monomers is an amino acid residue. Preferred polymers are
polymer molecules selected from the group consisting of dendrimers
as disclosed in PCT/DK2004/000531, polyalkylene oxide (PAO),
including polyalkylene glycol (PAG), such as polyethylene glycol
(PEG) and polypropylene glycol (PPG), branched PEGs, polyvinyl
alcohol (PVA), polycarboxylate, poly-vinylpyrolidone,
polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid
anhydride, and dextran, including carboxymethyl-dextran, PEG being
particularly preferred.
[0027] The term "PEGylated IL-21" means IL-21, having one or more
PEG molecule conjugated to a human IL-21 polypeptide. It is to be
understood, that the PEG molecule may be attached to any part of
the IL-21 polypeptide including any amino acid residue or
carbohydrate moiety of the IL-21 polypeptide. The term
"cysteine-PEGylated IL-21" means IL-21 having a PEG molecule
conjugated to a sulfhydryl group of a cysteine introduced in
IL-21.
[0028] The term "polyethylene glycol" or "PEG" means a polyethylene
glycol compound or a derivative thereof, with or without coupling
agents, coupling or activating moeities (e.g., with thiol,
triflate, tresylate, azirdine, oxirane, or preferably with a
maleimide moiety). Compounds such as maleimido monomethoxy PEG are
exemplary of activated PEG compounds of the invention. The term
"PEG" is intended to indicate polyethylene glycol of a molecular
weight between 500 and 150,000 Da, including analogues thereof,
wherein for instance the terminal OH-group has been replaced by a
methoxy group (referred to as mPEG).
[0029] In the present context, the words "peptide" and
"polypeptide" and "protein" are used interchangeably and are
intended to indicate the same.
[0030] In the context of the present invention "treatment" or
"treating" refers to preventing, alleviating, managing, curing or
reducing the disease e.g. a symptom of he disease, a condition
underlying the disease or both.
[0031] The term "functional in vivo half-life" is used in its
normal meaning, i.e., the time at which 50% of the biological
activity of the polypeptide or conjugate is still present in the
body/target organ, or the time at which the activity of the
polypeptide or conjugate is 50% of its initial value. As an
alternative to determining functional in vivo half-life, "serum
half-life" may be determined, i.e., the time at which 50% of the
polypeptide or conjugate molecules circulate in the plasma or
bloodstream prior to being cleared. Determination of
serum-half-life is often more simple than determining functional
half-life and the magnitude of serum-half-life is usually a good
indication of the magnitude of functional in vivo half-life.
Alternative terms to serum half-life include plasma half-life,
circulating half-life, circulatory half-life, serum clearance,
plasma clearance, and clearance half-life. The functionality to be
retained is normally selected from procoagulant, proteolytic,
co-factor binding, receptor binding activity, or other type of
biological activity associated with the particular protein.
[0032] The term "increased" with respect to the functional in vivo
half-life or plasma half-life is used to indicate that the relevant
half-life of the polypeptide or conjugate is statistically
significantly increased relative to that of a reference molecule,
such as non-conjugated glycoprotein as determined under comparable
conditions. For instance the relevant half-life may be increased by
at least about 25%, such as by at least about 50%, e.g., by at
least about 100%, 150%, 200%, 250%, or 500%.
[0033] In one embodiment the present invention relates to a use of
a derivative of IL-21 for the preparation of a medicament for the
treatment of diseases responsive to stimulation of T cell and NK
cell proliferation.
[0034] The present invention further provides a variety of other
polypeptide fusions [and related multimeric proteins comprising one
or more polypeptide fusions]. For example, a IL-21 polypeptide can
be prepared as a fusion to a dimerizing protein as disclosed in
U.S. Pat. Nos. 5,155,027 and 5,567,584 and derivatised according to
the invention. Preferred dimerizing proteins in this regard include
immunoglobulin constant region domains. Immunoglobulin-IL-21
polypeptide fusions can be expressed in genetically engineered
cells. Auxiliary domains can be fused to IL-21 polypeptides to
target them to specific cells, tissues, or macromolecules (e.g.,
collagen).
[0035] The petides of the present invention, including full-length
peptides, peptide fragments (e.g. ligand-binding fragments), and
fusion polypeptides can be produced in genetically engineered host
cells according to conventional techniques. Suitable host cells are
those cell types that can be transformed or transfected with
exogenous DNA and grown in culture, and include bacteria, fungal
cells, and cultured higher eukaryotic cells. Eukaryotic cells,
particularly cultured cells of multicellular organisms, are
preferred. Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are
disclosed by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed.(Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989), and Ausubel et al., ibid.
[0036] It is to be recognized that according to the present
invention, when a cDNA is claimed as described above, it is
understood that what is claimed are both the sense strand, the
anti-sense strand, and the DNA as double-stranded having both the
sense and anti-sense strand annealed together by their respective
hydrogen bonds. Also claimed is the messenger RNA (mRNA) which
encodes the polypeptides of the present invention, and which mRNA
is encoded by the above-described cDNA. A messenger RNA (mRNA) will
encode a polypeptide using the same codons as those defined above,
with the exception that each thymine nucleotide (T) is replaced by
a uracil nucleotide (U).
[0037] To direct an IL-21 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of the
protein, or may be derived from another secreted protein (e.g.,) or
synthesized de novo. The secretory signal sequence is joined to the
IL-21 DNA sequence in the correct reading frame. Secretory signal
sequences are commonly positioned 5' to the DNA sequence encoding
the polypeptide of interest, although certain signal sequences may
be positioned elsewhere in the DNA sequence of interest (see, e.
g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S.
Pat. No. 5,143,830).
[0038] IL-21 and variants thereof may be expressed in E-coli as
described in WO 04/55168. Optionally IL-21 variants may be produced
by recombinant DNA techniques in other organismes. To this end, DNA
sequences encoding human IL-21 related polypeptides or IL-21
variants may be isolated by preparing a genomic or cDNA library and
screening for DNA sequences coding for all or part of the protein
by hybridization using synthetic oligonucleotide probes in
accordance with standard techniques (cf. Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989). For the present purpose, the DNA
sequence encoding the protein is preferably of human origin, i.e.
derived from a human genomic DNA or cDNA library.
[0039] The DNA sequences encoding the IL-21 variants may also be
prepared synthetically by established standard methods, e.g. the
phosphoamidite method described by Beaucage and Caruthers,
Tetrahedron Letters 22 (1981), 1859-1869, or the method described
by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the
phosphoamidite method, oligonucleotides are synthesized, e.g. in an
automatic DNA synthesizer, purified, annealed, ligated and cloned
in suitable vectors.
[0040] The invention also comprises chemical modifications of the
IL-21 polypeptide, variant thereof or fusion proteins comprising
IL-21 or variants thereof. The chemical modification comprises
covalent modifications with an organic agent capable of reacting
with a selected side chain or a terminal residue.
[0041] Examples of such modifications are wherein a lipophilic
substituent is attached to one or more amino acid residues at a
position relative to the amino acid sequence of SEQ ID NO:1 or 2 as
described above. It is to be understood that an amino acid residues
at the position relative to the amino acid sequence of SEQ ID NO:2
may be any amino acid residue and not only the amino acid residue
naturally present at that position. In one embodiment the
lipophilic substituent is attached to a lysine.
[0042] One or more of the lysines in IL-21 could be derivatives as
described in the application. In other preferred embodiments,
additional lysines are substituted, inserted into the sequence or
added at the N-terminal or C-terminal, and then optionally
derivatised. Other aspects of the invention includes addition of
asp, glu, cys, gin, ser, thr, or tyr which carries function groups
in the side chain for derivatising.
[0043] Preferred regions of insertions are where the overall
activity of the protein is not adversely affected. N-terminal and
C-terminal truncations may occur simultaneously as well as
additions in the terminal of appropiate sequences.
[0044] In aspects of the invention any of the following positions
are selected for substitution optionally in combination: Lys22,
Lys53, Lys57, Lys76, Lys78, Lys89, Lys102, Lys103, Lys106, Lys113
or Lys118.
[0045] In an aspect of the invention the following substituents may
be derivatised, optionally in combination and optionally after
preparing variants with Lysine in the corresponding positions;
Arg66, Arg86, Arg87, Arg91, Arg111 or Arg127. All of the above
positions are calculated from the positions of the IL-21 peptide as
described in WO00/53761 without the initial N-terminal sequence of
28 amino acids.
[0046] The term "lipophilic substituent" is characterised by
comprising 4-40 carbon atoms and having a solubility in water at
20.degree. C. in the range from about 0.1 mg/100 ml water to about
250 mg/100 ml water, such as in the range from about 0.3 mg/100 ml
water to about 75 mg/100 ml water. For instance, octanoic acid (C8)
has a solubility in water at 20.degree. C. of 68 mg/100 ml,
decanoic acid (C10) has a solubility in water at 20.degree. C. of
15 mg/100 ml, and octadecanoic acid (C18) has a solubility in water
at 20.degree. C. of 0.3 mg/100 ml.
[0047] To obtain a satisfactory protracted profile of action of the
IL-21 derivative, the lipophilic substituent attached to the IL-21
moiety, as an example comprises 4-40 carbon atoms, such as 8-25
carbon atoms. The lipophilic substituent may be attached to an
amino group of the IL-21 moiety by means of a carboxyl group of the
lipophilic substituent which forms an amide bond with an amino
group of the amino acid to which it is attached. As an alternative,
the lipophilic substituent may be attached to said amino acid in
such a way that an amino group of the lipophilic substituent forms
an amide bond with a carboxyl group of the amino acid. As a further
option, the lipophililic substituent may be linked to the IL-21
moiety via an ester bond. Formally, the ester can be formed either
by reaction between a carboxyl group of the IL-21 moiety and a
hydroxyl group of the substituent-to-be or by reaction between a
hydroxyl group of the IL-21 moiety and a carboxyl group of the
substituent-to-be. As a further alternative, the lipophilic
substituent can be an alkyl group which is introduced into a
primary amino group of the IL-21 moiety
[0048] In one embodiment of the invention the IL-21 derivative only
has one lipophilic substituent attached to the IL-21 peptide.
[0049] In one embodiment of the invention the lipophilic
substituent comprises from 4 to 40 carbon atoms.
[0050] In one embodiment of the invention the lipophilic
substituent comprises from 8 to 25 carbon atoms.
[0051] In one embodiment of the invention the lipophilic
substituent comprises from 12 to 20 carbon atoms.
[0052] In one embodiment of the invention the lipophilic
substituent is attached to an amino acid residue in such a way that
a carboxyl group of the lipophilic substituent forms an amide bond
with an amino group of the amino acid residue.
[0053] In other preferred embodiments, additional lysines are
substituted, inserted into the sequence or added at the N-terminal
or C-terminal, and then optionally derivatised.
[0054] Preferred regions of insertions are where the overall
activity of the protein is not adversely affected. Preferred
regions are the positions listed above.
[0055] In one embodiment of the invention the lipophilic
substituent is attached to an amino acid residue in such a way that
an amino group of the lipophilic substituent forms an amide bond
with a carboxyl group of the amino acid residue.
[0056] In one embodiment of the invention the lipophilic
substituent is attached to the IL-21 peptide by means of a
spacer.
[0057] In one embodiment of the invention the spacer is an
unbranched alkane a,co-dicarboxylic acid group having from 1 to 7
methylene groups, such as two methylene groups which spacer forms a
bridge between an amino group of the IL-21 peptide and an amino
group of the lipophilic substituent.
[0058] In one embodiment of the invention the spacer is an amino
acid residue except a Cys residue, or a dipeptide. Examples of
suitable spacers includes .beta.-alanine, gamma-aminobutyric acid
(GABA), .gamma.-glutamic acid, succinic acid, Lys, Glu or Asp, or a
dipeptide such as Gly-Lys. When the spacer is succinic acid, one
carboxyl group thereof may form an amide bond with an amino group
of the amino acid residue, and the other carboxyl group thereof may
form an amide bond with an amino group of the lipophilic
substituent. When the spacer is Lys, Glu or Asp, the carboxyl group
thereof may form an amide bond with an amino group of the amino
acid residue, and the amino group thereof may form an amide bond
with a carboxyl group of the lipophilic substituent. When Lys is
used as the spacer, a further spacer may in some instances be
inserted between the .epsilon.-amino group of Lys and the
lipophilic substituent. In one embodiment, such a further spacer is
succinic acid which forms an amide bond with the .epsilon.-amino
group of Lys and with an amino group present in the lipophilic
substituent. In another embodiment such a further spacer is Glu or
Asp which forms an amide bond with the .epsilon.-amino group of Lys
and another amide bond with a carboxyl group present in the
lipophilic substituent, that is, the lipophilic substituent is a
N.sup..epsilon.-acylated lysine residue.
[0059] In one embodiment of the invention the spacer is selected
from the list consisting of .beta.-alanine, gamma-aminobutyric acid
(GABA), .gamma.-glutamic acid, Lys, Asp, Glu, a dipeptide
containing Asp, a dipeptide containing Glu, or a dipeptide
containing Lys. In one embodiment of the invention the spacer is
.beta.-alanine. In one embodiment of the invention the spacer is
gamma-aminobutyric acid (GABA). In one embodiment of the invention
the spacer is .gamma.-glutamic acid.
[0060] In one embodiment of the invention a carboxyl group of the
parent IL-21 peptide forms an amide bond with an amino group of a
spacer, and the carboxyl group of the amino acid or dipeptide
spacer forms an amide bond with an amino group of the lipophilic
substituent.
[0061] In one embodiment of the invention an amino group of the
parent IL-21 peptide forms an amide bond with a carboxylic group of
a spacer, and an amino group of the spacer forms an amide bond with
a carboxyl group of the lipophilic substituent.
[0062] In one embodiment of the invention the lipophilic
substituent comprises a partially or completely hydrogenated
cyclopentanophenathrene skeleton.
[0063] In one embodiment of the invention the lipophilic
substituent is an straight-chain or branched alkyl group. In one
embodiment of the invention the lipophilic substituent is the acyl
group of a straight-chain or branched fatty acid.
[0064] In one embodiment of the invention the acyl group of a
lipophilic substituent is selected from the group comprising
CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 4 to 38, such as
CH.sub.3(CH.sub.2).sub.6CO--, CH.sub.3(CH.sub.2).sub.8CO--,
CH.sub.3(CH.sub.2).sub.10CO--, CH.sub.3(CH.sub.2).sub.12CO--,
CH.sub.3(CH.sub.2).sub.14CO--, CH.sub.3(CH.sub.2).sub.16CO--,
CH.sub.3(CH.sub.2).sub.18CO--, CH.sub.3(CH.sub.2).sub.20CO-- and
CH.sub.3(CH.sub.2).sub.22CO--.
[0065] In one embodiment of the invention the lipophilic
substituent is an acyl group of a straight-chain or branched alkane
.alpha.,.omega.-dicarboxylic acid.
[0066] In one embodiment of the invention the acyl group of the
lipophilic substituent is selected from the group comprising
HOOC(CH.sub.2).sub.mCO--, wherein m is 4 to 38, such as
HOOC(CH.sub.2).sub.14CO--, HOOC(CH.sub.2).sub.16CO--,
HOOC(CH.sub.2).sub.18CO--, HOOC(CH.sub.2).sub.20CO-- and
HOOC(CH.sub.2).sub.22CO--.
[0067] In one embodiment of the invention the lipophilic
substituent is a group of the formula
CH.sub.3(CH.sub.2).sub.p((CH.sub.2).sub.qCOOH)CHNH--CO(CH.sub.2).sub.2CO--
-, wherein p and q are integers and p+q is an integer of from 8 to
40, such as from 12 to 35.
[0068] In one embodiment of the invention the lipophlic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.rCO--NHCH(COOH)(CH.sub.2).sub.2CO--, wherein
r is an integer of from 10 to 24.
[0069] In one embodiment of the invention the lipophilic
substituent is a group of the formula
CH.sub.3(CH.sub.2).sub.sCO--NHCH((CH.sub.2).sub.2COOH)CO--, wherein
s is an integer of from 8 to 24.
[0070] In one embodiment of the invention the lipophilic
substituent is a group of the formula COOH(CH.sub.2).sub.tCO--
wherein t is an integer of from 8 to 24.
[0071] In one embodiment of the invention the lipophilic
substituent is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.uCH.sub.3, wherein
u is an integer of from 8 to 18.
[0072] In one embodiment of the invention the lipophilic
substituent is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--COCH((CH.sub.2).sub.2COOH)NH--CO(CH.sub.2-
).sub.wCH.sub.3, wherein w is an integer of from 10 to 16.
[0073] In one embodiment of the invention the lipophilic
substituent is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NH--CO(CH.sub.2-
).sub.xCH.sub.3, wherein x is an integer of from 10to 16.
[0074] In one embodiment of the invention the lipophilic
substituent is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NHCO(CH.sub.2).-
sub.yCH.sub.3, wherein y is zero or an integer of from 1 to 22.
[0075] In one embodiment of the invention the lipophilic
substituent is N-Lithocholoyl.
[0076] In one embodiment of the invention the lipophilic
substituent is N-Choloyl.
[0077] In one embodiment of the invention the IL-21 derivative has
one lipophilic substituent. In one embodiment of the invention the
IL-21 derivative has two lipophilic substituents. In one embodiment
of the invention the IL-21 derivative has three lipophilic
substituents. In one embodiment of the invention the IL-21
derivative has four lipophilic substituents.
[0078] The methods of the present invention also contemplate using
chemically modified IL-21 compositions, in which an IL-21
polypeptide is linked with a polymeric molecule. Illustrative IL-21
polypeptides are soluble polypeptides that lack a functional
transmembrane domain, such as a mature IL-21 polypeptide.
Typically, the polymer is water soluble so that the IL-21 conjugate
does not precipitate in an aqueous environment, such as a
physiological environment. An example of a suitable polymer is one
that has been modified to have a single reactive group, such as an
active ester for acylation, or an aldehyde for alkylation, In this
way, the degree of polymerization can be controlled. An example of
a reactive aldehyde is polyethylene glycol propionaldehyde, or
mono-(C1-C10) alkoxy, or aryloxy derivatives thereof (see, for
example, Harris, et al., U.S. Pat. No. 5,252,714). The polymer may
be branched or unbranched. Moreover, a mixture of polymers can be
used to produce IL-21 conjugates. IL-21 conjugates used for therapy
can comprise pharmaceutically acceptable water-soluble polymer
moieties. Suitable water-soluble polymers include polyethylene
glycol (PEG), monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG,
aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG,
PEG propionaldehyde, bis-succinimidyl carbonate PEG, propylene
glycol homopolymers, a polypropylene oxide/ethylene oxide
co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl
alcohol, dextran, cellulose, or other carbohydrate-based polymers.
Different sizes of PEG are described above.
[0079] In an aspect of the invention the peptide is derivatised
with a N-terminal PEG group by oxidation of a serine with sodium
periodate, followed by reaction of PEG-derivative, to which a
hydroxylamine was attached, yielding an oxime. In principle, the
serine could also have been an internal serine residue or an added
serine residue as described above.
[0080] Other methods for attaching PEG groups are described in G.
Pasut, A. Guiotto, F. M. Veronese Expert Opin. Ther. Patents 2004,
14, 859-894. Variants of IL-21 suitable for attachment of polymeric
groups may be obtained as described above.
[0081] In an aspect of the invention IL-21 is attached in the
C-terminal of the peptide. This may be achieved by using IL-21 or a
variant of IL-21 suitable as a substrate for CPY (carboxypeptidase
Y) of which part of the reaction is described in in EP243929. This
intermediate may then be further substituted by a compound
containing one or more reactive groups, X, suitable for further
substitution of with molecules containing the reactive group Y. The
reactive group may be selected from the groups mentioned below.
[0082] In one embodiment the functional groups of X and Y are
selected from amongst carbonyl groups, such as keto and aldehyde
groups, and amino derivatives, such as TABLE-US-00001 hydrazine
derivatives --NH--NH.sub.2, hydrazine carboxylate
--O--C(O)--NH--NH.sub.2, derivatives semicarbazide derivatives
--NH--C(O)--NH--NH.sub.2, thiosemicarbazide
--NH--C(S)--NH--NH.sub.2, derivatives carbonic acid dihydrazide
--NHC(O)--NH--NH--C(O)--NH--NH.sub.2, derivatives carbazide
derivatives --NH--NH--C(O)--NH--NH.sub.2, thiocarbazide derivatives
--NH--NH--C(S)--NH--NH.sub.2, aryl hydrazine derivatives
--NH--C(O)--C.sub.6H.sub.4--NH--NH.sub.2, and hydrazide derivatives
--C(O)--NH--NH.sub.2;
oxylamine derivatives, such as --O--NH.sub.2, --C(O)--O--NH.sub.2,
--NH--C(O)--O--NH.sub.2 and --NH--C(S)--O--NH.sub.2.
[0083] It is to be understood, that if the functional group
comprised in X is a carbonyl group, then the functional group
comprised in Y is an amine derivative, and vice versa. Due to the
presence of --NH.sub.2 groups in most peptides, a better
selectivity is believed to be obtained if X comprises a keto- or an
aldehyde-functionality.
[0084] Examples of derivatives of PEG suitable in the reaction
described above are ##STR2##
[0085] wherein n is 1,2, 3, 4, 5 or 6 and mPEG has a molecular
weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa. ##STR3##
[0086] wherein m is 1, 2, 3, 4, 5 or 6 and mPEG has a molecular
weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa. ##STR4##
[0087] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR5##
[0088] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR6##
[0089] wherein n is 0,1,2,3,4,5 or 6 and m is 1, 2,3, 4, 5 or 6 and
mPEG has a molecular weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa,
##STR7##
[0090] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR8##
[0091] wherein n is 1, 2, 3, 4, 5 or 6 and mPEG has a molecular
weight of 10 kDa, 20 kDa, 30 kDa or 40 kDa, ##STR9##
[0092] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR10##
[0093] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR11##
[0094] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR12##
[0095] wherein mPEG has a molecular weight of 10 kDa, 20 kDa, 30
kDa or 40 kDa, ##STR13##
[0096] wherein Y is --O--NH.sub.2, NH--NH.sub.2,
[0097] n, m and s are any number from 0 to 20;
[0098] R' and R'' independently represents for example methyl,
phenyl, biphenyl, phenoxyphenyl, phenylcarboxyphenyl.
[0099] At any suitable position in the alkyl chains in any of the
formulas above a group of the formula --SO.sub.2--, --C(O)NH--,
--C(O)NHSO.sub.2--, --SO.sub.2-phenyl-, C(O)NHSO.sub.2-phenyl- may
be inserted in either direction. Optionally the group C(O)NH in the
above formula may be substituted by ##STR14##
[0100] In an embodiment of the invention the introduction of the
derivative is introduced in one step. The R--X then contains the
derivatives to be introduced into IL-21. The nucleophile represents
for example amino acids, which has been modified to carry the
derivative. In principle any sequence of amino acids may be used.
In an aspect of the invention nucleophiles such as G(.sub.1-5)-PEG,
G(.sub.1-5)-lipid. G(.sub.1-4) --NH--CH.sub.2--CHO, G(.sub.1-4)
--NH--CH.sub.2--C--O--NH.sub.2 etc. are used.
[0101] PEG is a suitable polymer molecule, since it has only few
reactive groups capable of cross-linking compared to
polysaccharides such as dextran. In particular, monofunctional PEG,
e.g. methoxypolyethylene glycol (mPEG), is of interest since its
coupling chemistry is relatively simple (only one reactive group is
available for conjugating with attachment groups on the
polypeptide). Consequently, the risk of cross-linking is
eliminated, the resulting polypeptide conjugates are more
homogeneous and the reaction of the polymer molecules with the
polypeptide is easier to control.
[0102] To effect covalent attachment of the polymer molecule(s) to
the polypeptide, the hydroxyl end groups of the polymer molecule
are provided in activated form, i.e. with reactive functional
groups. Suitable activated polymer molecules are commercially
available, e.g. from Shearwater Corp., Huntsville, Ala., USA, or
from PolyMASC Pharmaceuticals pic, UK. Alternatively, the polymer
molecules can be activated by conventional methods known in the
art, e.g. as disclosed in WO 90/13540. Specific examples of
activated linear or branched polymer molecules for use in the
present invention are described in the Shearwater Corp. 1997 and
2000 Catalogs (Functionalized Biocompatible Polymers for Research
and pharmaceuticals, Polyethylene Glycol and Derivatives,
incorporated herein by reference). Specific examples of activated
PEG polymers include the following linear PEGs: NHS-PEG (e.g.
SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and
SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,
CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and
branched PEGs such as PEG2-NHS and those disclosed in U.S. Pat. No.
5,932,462 and U.S. Pat. No. 5,643,575, both of which are
incorporated herein by reference. Furthermore, the following
publications, incorporated herein by reference, disclose useful
polymer molecules and/or PEGylation chemistries: U.S. Pat. No.
5,824,778, U.S. Pat. No. 5,476,653, WO 97/32607, EP 229,108, EP
402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No. 5,281,698, U.S.
Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO 92/16555, WO
94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO
95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO 96/00080, WO
97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO
99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO
97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.
5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO
96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No.
5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400
472, EP 183 503 and EP 154 316.
[0103] The conjugation of the polypeptide and the activated polymer
molecules is conducted by use of any conventional method, e.g. as
described in the following references (which also describe suitable
methods for activation of polymer molecules): R. F. Taylor, (1991),
"Protein immobilisation. Fundamental and applications", Marcel
Dekker, N.Y.; S. S. Wong, (1992), "Chemistry of Protein Conjugation
and Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al.,
(1993), "Immobilized Affinity Ligand Techniques", Academic Press,
N.Y.). The skilled person will be aware that the activation method
and/or conjugation chemistry to be used depends on the attachment
group(s) of the polypeptide (examples of which are given further
above), as well as the functional groups of the polymer (e.g. being
amine, hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl,
maleimide, vinysulfone or haloacetate). The PEG-ylation may be
directed towards conjugation to all available attachment groups on
the polypeptide (i.e. such attachment groups that are exposed at
the surface of the polypeptide) or may be directed towards one or
more specific attachment groups, e.g. the N-terminal amino group
(U.S. Pat. No. 5,985,265). Furthermore, the conjugation may be
achieved in one step or in a stepwise manner (e.g. as described in
WO 99/55377).
[0104] It will be understood that the PEGylation is designed so as
to produce the optimal molecule with respect to the number of PEG
molecules attached, the size and form of such molecules (e.g.
whether they are linear or branched), and where in the polypeptide
such molecules are attached. The molecular weight of the polymer to
be used will be chosen taking into consideration the desired effect
to be achieved. For instance, if the primary purpose of the
conjugation is to achieve a conjugate having a high molecular
weight and larger size (e.g. to reduce renal clearance), one may
choose to conjugate either one or a few high molecular weight
polymer molecules or a number of polymer molecules with a smaller
molecular weight to obtain the desired effect. Preferably, however,
several polymer molecules with a lower molecular weight will be
used. This is also the case if a high degree of epitope shielding
is desired. In such cases, 2-8 polymers with a molecular weight of
e.g. about 5,000 Da, such as 3-6 such polymers, may for example be
used. As the examples below illustrate, it may be advantageous to
have a larger number of polymer molecules with a lower molecular
weight (e.g. 4-6 with a MW of 5000) compared to a smaller number of
polymer molecules with a higher molecular weight (e.g. 1-3 with a
MW of 12,000-20,000) in terms of improving the functional in vivo
half-life of the polypeptide conjugate, even where the total
molecular weight of the attached polymer molecules in the two cases
is the same or similar. It is believed that the presence of a
larger number of smaller polymer molecules provides the polypeptide
with a larger diameter or apparent size than e.g. a single yet
larger polymer molecule, at least when the polymer molecules are
relatively uniformly distributed on the polypeptide surface. It has
further been found that advantageous results are obtained when the
apparent size (also referred to as the "apparent molecular weight"
or "apparent mass") of at least a major portion of the conjugate of
the invention is at least about 50 kDa, such as at least about 55
kDa, such as at least about 60 kDa, e.g. at least about 66 kDa.
This is believed to be due to the fact that renal clearance is
substantially eliminated for conjugates having a sufficiently large
apparent size. In the present context, the "apparent size" of a
IL-21 conjugate or IL-21 polypeptide is determined by the SDS-PAGE
method.
[0105] In an embodiment of the invention PEG is conjugated to a
peptide according to the present invention may be of any molecular
weight. In particular the molecular weight may be between 500 and
100,000 Da, such as between 500 and 60,000 Da, such as between 1000
and 40,000 Da, such as between 5,000 and 40,000 Da. In particular,
PEG with molecular weights of 10,000 Da, 20,000 Da or 40,000 KDa
may be used in the present invention. In all cases the PEGs may be
linear or branched. In an embodiment of the invention the PEG
groups are 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa og 60 kDa.
[0106] In an embodiment of the invention, one or more polymeric
molecules are added to the peptide.
[0107] The present invention provides compounds which are suitable
for attachment of a polymeric group. In an embodiment of the
invention the derivative thus provides a peptide which has an
improved in vivo half life. This may be achieved by protecting the
compound against chemical degradation, proteolytic degradation, or
antibody recognition--or any other mechanisms.
[0108] In an embodiment the compounds provided are less toxic.
[0109] In an embodiment the compounds provided are more water
soluble
[0110] In an embodiment the compounds provided has a modified
biodistribution.
[0111] The above are with reference to the non-derivatised
analogues or to the hIL-21.
Pharmaceutical Compositions
[0112] The invention provides in a particular embodiment the
following:
[0113] Another object of the present invention is to provide a
pharmaceutical formulation comprising IL-21, analogues or
derivatives thereof, or optionally together with any other compound
mentioned in the present application which is present in a
concentration from 0.1 mg/ml to 100 mg/ml, and wherein said
formulation has a pH from 2.0 to 10.0. The formulation may further
comprise a buffer system, preservative(s), tonicity agent(s),
chelating agent(s), stabilizers and surfactants. In one embodiment
of the invention the pharmaceutical formulation is an aqueous
formulation, i.e. formulation comprising water. Such formulation is
typically a solution or a suspension. In a further embodiment of
the invention the pharmaceutical formulation is an aqueous
solution. The term "aqueous formulation" is defined as a
formulation comprising at least 50% w/w water. Likewise, the term
"aqueous solution" is defined as a solution comprising at least 50%
w/w water, and the term "aqueous suspension" is defined as a
suspension comprising at least 50% w/w water.
[0114] In another embodiment the pharmaceutical formulation is a
freeze-dried formulation, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0115] In another embodiment the pharmaceutical formulation is a
dried formulation (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0116] In a further aspect the invention relates to a
pharmaceutical formulation comprising an aqueous solution of IL-21
or any other compound as mentioned above and a buffer, wherein said
compound is present in a concentration from 0.1 mg/ml or as
mentioned above, preferably from 0.5 mg/ml-50 mg/ml and wherein
said formulation has a pH from about 2.0 to about 10.0. Preferred
pH is from 3.0 to about 8.0. Particular preferred range is from
4.0-6.0, such as for example the ranges 4.0-4.5, 4.5-5.0, 5.0-5.5
and 5.5-6.0.
[0117] In another embodiment of the invention the pH of the
formulation is selected from the list consisting of 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and
10.0.
[0118] In a further embodiment of the invention the buffer is
selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine,
arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate,
sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine,
tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric
acid, aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0119] In a further embodiment of the invention the formulation
further comprises a pharmaceutically acceptable antimicrobial
preservative. In a further embodiment of the invention the
preservative is selected from the group consisting of phenol,
o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl
p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,
2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomersal,
bronopol, benzoic acid, imidurea, chlorohexidine, sodium
dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium
chloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or
mixtures thereof. In a further embodiment of the invention the
preservative is present in a concentration from 0.1 mg/ml to 20
mg/ml. In a further embodiment of the invention the preservative is
present in a concentration from 0.1 mg/ml to 5 mg/ml. In a further
embodiment of the invention the preservative is present in a
concentration from 5 mg/ml to 10 mg/ml. In a further embodiment of
the invention the preservative is present in a concentration from
10 mg/ml to 20 mg/ml. Each one of these specific preservatives
constitutes an alternative embodiment of the invention. The use of
a preservative in pharmaceutical compositions is well-known to the
skilled person. For convenience reference is made to Remington: The
Science and Practice of Pharmacy, 19.sup.th edition, 1995.
[0120] In a further embodiment of the invention the formulation
further comprises an isotonic agent. In a further embodiment of the
invention the isotonic agent is selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. L-glycine, L-histidine, arginine, lysine,
isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g.
glycerol (glycerine), 1,2-propanediol (propyleneglycol),
1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG400),
or mixtures thereof. Any sugar such as mono-, di-, or
polysaccharides, or water-soluble glucans, including for example
fructose, glucose, mannose, sorbose, xylose, maltose, lactose,
sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,
soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na
may be used. In one embodiment the sugar additive is sucrose. Sugar
alcohol is defined as a C4-C8 hydrocarbon having at least one --OH
group and includes, for example, mannitol, sorbitol, inositol,
galactitol, dulcitol, xylitol, and arabitol. In one embodiment the
sugar alcohol additive is mannitol. The sugars or sugar alcohols
mentioned above may be used individually or in combination. There
is no fixed limit to the amount used, as long as the sugar or sugar
alcohol is soluble in the liquid preparation and does not adversely
effect the stabilizing effects achieved using the methods of the
invention. In one embodiment, the sugar or sugar alcohol
concentration is between about 1 mg/ml and about 150 mg/ml. In a
further embodiment of the invention the isotonic agent is present
in a concentration from 1 mg/ml to 50 mg/ml. In a further
embodiment of the invention the isotonic agent is present in a
concentration from 1 mg/ml to 7 mg/ml. In a further embodiment of
the invention the isotonic agent is present in a concentration from
8 mg/ml to 24 mg/ml. In a further embodiment of the invention the
isotonic agent is present in a concentration from 25 mg/ml to 50
mg/ml. Each one of these specific isotonic agents constitutes an
alternative embodiment of the invention. The use of an isotonic
agent in pharmaceutical compositions is well-known to the skilled
person. For convenience reference is made to Remington: The Science
and Practice of Pharmacy, 19.sup.th edition, 1995.
[0121] In a further embodiment of the invention the formulation
further comprises a chelating agent. In a further embodiment of the
invention the chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. In a further embodiment of the
invention the chelating agent is present in a concentration from
0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the
chelating agent is present in a concentration from 0.1 mg/ml to 2
mg/ml. In a further embodiment of the invention the chelating agent
is present in a concentration from 2 mg/ml to 5 mg/ml. Each one of
these specific chelating agents constitutes an alternative
embodiment of the invention. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0122] In a further embodiment of the invention the formulation
further comprises a stabilizer. The use of a stabilizer in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0123] More particularly, compositions of the invention are
stabilized liquid pharmaceutical compositions whose therapeutically
active components include a polypeptide that possibly exhibits
aggregate formation during storage in liquid pharmaceutical
formulations. By "aggregate formation" is intended a physical
interaction between the polypeptide molecules that results in
formation of oligomers, which may remain soluble, or large visible
aggregates that precipitate from the solution. By "during storage"
is intended a liquid pharmaceutical composition or formulation once
prepared, is not immediately administered to a subject. Rather,
following preparation, it is packaged for storage, either in a
liquid form, in a frozen state, or in a dried form for later
reconstitution into a liquid form or other form suitable for
administration to a subject. By "dried form" is intended the liquid
pharmaceutical composition or formulation is dried either by freeze
drying (i.e., lyophilization; see, for example, Williams and Polli
(1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see
Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific
and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992)
Drug Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994)
Pharm. Res. 11:12-20), or air drying (Carpenter and Crowe (1988)
Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Aggregate formation by a polypeptide during storage of a liquid
pharmaceutical composition can adversely affect biological activity
of that polypeptide, resulting in loss of therapeutic efficacy of
the pharmaceutical composition. Furthermore, aggregate formation
may cause other problems such as blockage of tubing, membranes, or
pumps when the polypeptide-containing pharmaceutical composition is
administered using an infusion system.
[0124] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease
aggregate formation by the polypeptide during storage of the
composition. By "amino acid base" is intended an amino acid or a
combination of amino acids, where any given amino acid is present
either in its free base form or in its salt form. Where a
combination of amino acids is used, all of the amino acids may be
present in their free base forms, all may be present in their salt
forms, or some may be present in their free base forms while others
are present in their salt forms. In one embodiment, amino acids to
use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic
acid, and glutamic acid. Any stereoisomer (i.e., L, D, or DL
isomer) of a particular amino acid (e.g. glycine, methionine,
histidine, imidazole, arginine, lysine, isoleucine, aspartic acid,
tryptophan, threonine and mixtures thereof) or combinations of
these stereoisomers, may be present in the pharmaceutical
compositions of the invention so long as the particular amino acid
is present either in its free base form or its salt form. In one
embodiment the L-stereoisomer is used. Compositions of the
invention may also be formulated with analogues of these amino
acids. By "amino acid analogue" is intended a derivative of the
naturally occurring amino acid that brings about the desired effect
of decreasing aggregate formation by the polypeptide during storage
of the liquid pharmaceutical compositions of the invention.
Suitable arginine analogues include, for example, aminoguanidine,
ornithine and N-monoethyl L-arginine, suitable methionine analogues
include ethionine and buthionine and suitable cysteine analogues
include S-methyl-L cysteine. As with the other amino acids, the
amino acid analogues are incorporated into the compositions in
either their free base form or their salt form. In a further
embodiment of the invention the amino acids or amino acid analogues
are used in a concentration, which is sufficient to prevent or
delay aggregation of the protein.
[0125] In a further embodiment of the invention methionine (or
other sulphuric amino acids or amino acid analogous) may be added
to inhibit oxidation of methionine residues to methionine sulfoxide
when the polypeptide acting as the therapeutic agent is a
polypeptide comprising at least one methionine residue susceptible
to such oxidation. By "inhibit" is intended minimal accumulation of
methionine oxidized species over time. Inhibiting methionine
oxidation results in greater retention of the polypeptide in its
proper molecular form. Any stereoisomer of methionine (L, D, or DL
isomer) or combinations thereof can be used. The amount to be added
should be an amount sufficient to inhibit oxidation of the
methionine residues such that the amount of methionine sulfoxide is
acceptable to regulatory agencies. Typically, this means that the
composition contains no more than about 10% to about 30% methionine
sulfoxide. Generally, this can be achieved by adding methionine
such that the ratio of methionine added to methionine residues
ranges from about 1:1 to about 1000:1, such as 10:1 to about
100:1.
[0126] In a further embodiment of the invention the formulation
further comprises a stabilizer selected from the group of high
molecular weight polymers or low molecular compounds. In a further
embodiment of the invention the stabilizer is selected from
polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA),
polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates
thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,
sulphur-containing substances as monothioglycerol, thioglycolic
acid and 2-methylthioethanol, and different salts (e.g. sodium
chloride). Each one of these specific stabilizers constitutes an
alternative embodiment of the invention.
[0127] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a
therapeutically active polypeptide therein. Stabilizing agents of
particular interest to the present invention include, but are not
limited to, methionine and EDTA, which protect the polypeptide
against methionine oxidation, and a nonionic surfactant, which
protects the polypeptide against aggregation associated with
freeze-thawing or mechanical shearing.
[0128] In a further embodiment of the invention the formulation
further comprises a surfactant. In a further embodiment of the
invention the surfactant is selected from a detergent, ethoxylated
castor oil, polyglycolyzed glycerides, acetylated monoglycerides,
sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block
polymers (eg. poloxamers such as Pluronic.RTM. F68, poloxamer 188
and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid
esters, polyoxyethylene and polyethylene derivatives such as
alkylated and alkoxylated derivatives (tweens, e.g. Tween-20,
Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated
derivatives thereof, diglycerides or polyoxyethylene derivatives
thereof, alcohols, glycerol, lectins and phospholipids (eg.
phosphatidyl serine, phosphatidyl choline, phosphatidyl
ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and
sphingomyelin), derivates of phospholipids (eg. dipalmitoyl
phosphatidic acid) and lysophospholipids (eg. palmitoyl
lysophosphatidyl-L-serine and 1-acyl-sn-glycero-3-phosphate esters
of ethanolamine, choline, serine or threonine) and alkyl, alkoxyl
(alkyl ester), alkoxy (alkyl ether)-derivatives of lysophosphatidyl
and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of
lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and
modifications of the polar head group, that is cholines,
ethanolamines, phosphatidic acid, serines, threonines, glycerol,
inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP,
lysophosphatidylserine and lysophosphatidylthreonine, and
glycerophospholipids (eg. cephalins), glyceroglycolipids (eg.
galactopyransoide), sphingoglycolipids (eg. ceramides,
gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic
acid derivatives-(e.g. sodium tauro-dihydrofusidate etc.),
long-chain fatty acids and salts thereof C6-C12 (eg. oleic acid and
caprylic acid), acylcarnitines and derivatives,
N.sup..alpha.-acylated derivatives of lysine, arginine or
histidine, or side-chain acylated derivatives of lysine or
arginine, N.sup..alpha.-acylated derivatives of dipeptides
comprising any combination of lysine, arginine or histidine and a
neutral or acidic amino acid, N.sup..alpha.-acylated derivative of
a tripeptide comprising any combination of a neutral amino acid and
two charged amino acids, DSS (docusate sodium, CAS registry no
[577-11-7]), docusate calcium, CAS registry no [128-49-4]),
docusate potassium, CAS registry no [7491-09-0]), SDS (sodium
dodecyl sulphate or sodium lauryl sulphate), sodium caprylate,
cholic acid or derivatives thereof, bile acids and salts thereof
and glycine or taurine conjugates, ursodeoxycholic acid, sodium
cholate, sodium deoxycholate, sodium taurocholate, sodium
glycocholate, N-Hexadecyl-N,
N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic
surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyidimethylammonio-1-propanesulfonate, cationic
surfactants (quaternary ammonium bases) (e.g.
cetyl-trimethylammonium bromide, cetylpyridinium chloride),
non-ionic surfactants (eg. Dodecyl-D-glucopyranoside), poloxamines
(eg. Tetronic's), which are tetrafunctional block copolymers
derived from sequential addition of propylene oxide and ethylene
oxide to ethylenediamine, or the surfactant may be selected from
the group of imidazoline derivatives, or mixtures thereof. Each one
of these specific surfactants constitutes an alternative embodiment
of the invention.
[0129] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0130] It is possible that other ingredients may be present in the
peptide pharmaceutical formulation of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers, chelating agents,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatine or proteins) and a zwitterion (e.g., an amino
acid such as betaine, taurine, arginine, glycine, lysine and
histidine). Such additional ingredients, of course, should not
adversely affect the overall stability of the pharmaceutical
formulation of the present invention.
[0131] Pharmaceutical compositions containing IL-21 or any other
compound as mentioned above according to the present invention may
be administered to a patient in need of such treatment at several
sites, for example, at topical sites, for example, skin and mucosal
sites, at sites which bypass absorption, for example,
administration in an artery, in a vein, in the heart, and at sites
which involve absorption, for example, administration in the skin,
under the skin, in a muscle or in the abdomen.
[0132] Administration of pharmaceutical compositions according to
the invention may be through several routes of administration, for
example, lingual, sublingual, buccal, in the mouth, oral, in the
stomach and intestine, nasal, pulmonary, for example, through the
bronchioles and alveoli or a combination thereof, epidermal,
dermal, transdermal, vaginal, rectal, ocular, for examples through
the conjunctiva, uretal, and parenteral to patients in need of such
a treatment.
[0133] Compositions of the current invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, rinses,
capsules, for example, hard gelatine capsules and soft gelatine
capsules, suppositories, rectal capsules, drops, gels, sprays,
powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal
ointments, injection solution, in situ transforming solutions, for
example in situ gelling, in situ setting, in situ precipitating, in
situ crystallization, infusion solution, and implants.
[0134] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of of IL-21 or any other compound as mentioned above,
increase bioavailability, increase solubility, decrease adverse
effects, achieve chronotherapy well known to those skilled in the
art, and increase patient compliance or any combination thereof.
Examples of carriers, drug delivery systems and advanced drug
delivery systems include, but are not limited to, polymers, for
example cellulose and derivatives, polysaccharides, for example
dextran and derivatives, starch and derivatives, poly(vinyl
alcohol), acrylate and methacrylate polymers, polylactic and
polyglycolic acid and block co-polymers thereof, polyethylene
glycols, carrier proteins, for example albumin, gels, for example,
thermogelling systems, for example block co-polymeric systems well
known to those skilled in the art, micelles, liposomes,
microspheres, nanoparticulates, liquid crystals and dispersions
thereof, L2 phase and dispersions there of, well known to those
skilled in the art of phase behaviour in lipid-water systems,
polymeric micelles, multiple emulsions, self-emulsifying,
self-microemulsifying, cyclodextrins and derivatives thereof, and
dendrimers.
[0135] Compositions of the current invention are useful in the
formulation of solids, semisolids, powder and solutions for
pulmonary administration of IL-21 or any other compound as
mentioned above using, for example a metered dose inhaler, dry
powder inhaler and a nebulizer, all being devices well known to
those skilled in the art.
[0136] Compositions of the current invention are specifically
useful in the formulation of controlled, sustained, protracting,
retarded, and slow release drug delivery systems. More
specifically, but not limited to, compositions are useful in
formulation of parenteral controlled release and sustained release
systems (both systems leading to a many-fold reduction in number of
administrations), well known to those skilled in the art. Even more
preferably, are controlled release and sustained release systems
administered subcutaneous. Without limiting the scope of the
invention, examples of useful controlled release system and
compositions are hydrogels, oleaginous gels, liquid crystals,
polymeric micelles, microspheres, nanoparticles,
[0137] Methods to produce controlled release systems useful for
compositions of the current invention include, but are not limited
to, crystallization, condensation, co-crystallization,
precipitation, co-precipitation, emulsification, dispersion, high
pressure homogenisation, encapsulation, spray drying,
microencapsulating, coacervation, phase separation, solvent
evaporation to produce microspheres, extrusion and supercritical
fluid processes. General reference is made to Handbook of
Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker,
New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99:
Protein Formulation and Delivery (MacNally, E. J., ed. Marcel
Dekker, New York, 2000).
[0138] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension for the administration of IL-21 or any other compound as
mentioned above, in the form of a nasal or pulmonal spray. As a
still further option, the pharmaceutical compositions containing
IL-21 or any other compound as mentioned above can also be adapted
to transdermal administration, e.g. by needle-free injection or
from a patch, optionally an iontophoretic patch, or transmucosal,
e.g. buccal, administration.
[0139] The term "stabilized formulation" refers to a formulation
with increased physical stability, increased chemical stability or
increased physical and chemical stability.
[0140] The term "physical stability" of the protein formulation as
used herein refers to the tendency of the protein to form
biologically inactive and/or insoluble aggregates of the protein as
a result of exposure of the protein to thermo-mechanical stresses
and/or interaction with interfaces and surfaces that are
destabilizing, such as hydrophobic surfaces and interfaces.
Physical stability of the aqueous protein formulations is evaluated
by means of visual inspection and/or turbidity measurements after
exposing the formulation filled in suitable containers (e.g.
cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual
inspection of the formulations is performed in a sharp focused
light with a dark background. The turbidity of the formulation is
characterized by a visual score ranking the degree of turbidity for
instance on a scale from 0 to 3 (a formulation showing no turbidity
corresponds to a visual score 0, and a formulation showing visual
turbidity in daylight corresponds to visual score 3). A formulation
is classified physically unstable with respect to protein
aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the formulation can be evaluated by
simple turbidity measurements well-known to the skilled person.
Physical stability of the aqueous protein formulations can also be
evaluated by using a spectroscopic agent or probe of the
conformational status of the protein. The probe is preferably a
small molecule that preferentially binds to a non-native conformer
of the protein. One example of a small molecular spectroscopic
probe of protein structure is Thioflavin T. Thioflavin T is a
fluorescent dye that has been widely used for the detection of
amyloid fibrils. In the presence of fibrils, and perhaps other
protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about
482 nm when bound to a fibril protein form. Unbound Thioflavin T is
essentially non-fluorescent at the wavelengths.
[0141] Other small molecules can be used as probes of the changes
in protein structure from native to non-native states. For instance
the "hydrophobic patch" probes that bind preferentially to exposed
hydrophobic patches of a protein. The hydrophobic patches are
generally buried within the tertiary structure of a protein in its
native state, but become exposed as a protein begins to unfold or
denature. Examples of these small molecular, spectroscopic probes
are aromatic, hydrophobic dyes, such as antrhacene, acridine,
phenanthroline or the like. Other spectroscopic probes are
metal-amino acid complexes, such as cobalt metal complexes of
hydrophobic amino acids, such as phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.
[0142] The term "chemical stability" of the protein formulation as
used herein refers to chemical covalent changes in the protein
structure leading to formation of chemical degradation products
with potential less biological potency and/or potential increased
immunogenic properties compared to the native protein structure.
Various chemical degradation products can be formed depending on
the type and nature of the native protein and the environment to
which the protein is exposed. Elimination of chemical degradation
can most probably not be completely avoided and increasing amounts
of chemical degradation products is often seen during storage and
use of the protein formulation as well-known by the person skilled
in the art. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl
residues is hydrolysed to form a free carboxylic acid. Other
degradation pathways involves formation of high molecular weight
transformation products where two or more protein molecules are
covalently bound to each other through transamidation and/or
disulfide interactions leading to formation of covalently bound
dimer, oligomer and polymer degradation products (Stability of
Protein Pharmaceuticals, Ahern. T. J. & Manning M. C., Plenum
Press, New York 1992). Oxidation (of for instance methionine
residues) can be mentioned as another variant of chemical
degradation. The chemical stability of the protein formulation can
be evaluated by measuring the amount of the chemical degradation
products at various time-points after exposure to different
environmental conditions (the formation of degradation products can
often be accelerated by for instance increasing temperature). The
amount of each individual degradation product is often determined
by separation of the degradation products depending on molecule
size and/or charge using various chromatography techniques (e.g.
SEC-HPLC and/or RP-HPLC).
[0143] Hence, as outlined above, a "stabilized formulation" refers
to a formulation with increased physical stability, increased
chemical stability or increased physical and chemical stability. In
general, a formulation must be stable during use and storage (in
compliance with recommended use and storage conditions) until the
expiration date is reached.
EXAMPLES
[0144] Recombinant Interleukin 21 (IL21) was expressed as inclusion
bodies in E.coli as described in WO04/55168 with a N-terminal
extension (Met-Ser-hIL21). The N-terminal Met residue is removed by
the protease systems present in E.coli, leaving Ser-hIL21. The
Glu-Ala-Glu amino acid sequence can be present or absent.
[0145] The protein was refolded and purified to 90-95% purity using
conventional chromatographic methods.
[0146] The pure protein was subsequently N-terminally PEGylated via
oxidation of the N-terminal serine by reaction with sodium
periodate, followed by reaction of PEG-derivative, to which a
hydroxylamine was attached, yielding an oxime.
[0147] Subsequent purification was done using gelfiltration or size
exclusion chromatography (SEC).
[0148] In an proliferation assay as for example the BAF3 assay
described below, the pegylated IL21 was equipotent with the
unpegylated standard, indicating that the pegylation does not
interfere with receptor binding, and that the reaction procedure
are not harmful to the protein.
PREPARATORY EXAMPLES
[0149] A method for the attachment of a PEG-moiety to the
C-terminus of a IL-21 derivative may be performed analogously to
the attachment of chemical moieties to other peptides or proteins
described above:
[0150] A PEG-moiety may be attached to the C-terminus of IL-21 or
an IL-21 derivative such as e.g. hIL-21, by a two step method.
[0151] In the first step, a suitable IL-21-analogue such as e.g.
(hIL-21yl)alanine is subjected to a transpeptidation reaction
catalyzed by carboxypeptidase Y (CPY) in the presence of a suitable
nucleophile e.g. (S)-2-amino-3-(4-(propargyloxy)phenyl)propanoic
acid in a suitable buffer such as a HEPES/TMEDA-buffer at a
suitable pH such e.g. pH 7.5 or pH8 at a suitable temperature such
as e.g. room temperature 30.degree. C. or 35.degree. C.
##STR15##
[0152] In the second step, a suitable derivatized PEG-reagent may
be reacted with
(S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide.
E.g. an excess of 4-(mPEG20000yl)-N-(3-(hydroxyimino)benzyl)
butanoic amide may be reacted under oxidative conditions such as
e.g. sodium hypochlorite solution to
4-(mPEG20000yl)-N-3-(oxycyano)benzylbutanoic amide. A solution of
4-(mPEG20000yl)-N-(3-(oxycyano)benzylbutanoic amide may be added to
a solution of
(S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide to
yield
(S)-2-((hIL-21yl)amino)-3-(4-((3-(3-((4-(mPEG20000yl)butanoylamino)methyl-
)phenyl)isoxazol-5-yl)methoxy)phenyl)propanoic amide. ##STR16##
[0153] 4-(mPEG20000yl)-N-3-(hydroxyimino)benzyl) butanoic amide may
be prepared from commercially available
3-((tert-butoxycarbonylamino)methyl)benzoic acid, which may be
reduced with a suitable reagent or combination of reagents e.g. in
a two step procedure known to a person trained in the art,
comprising in a first step addition of ethyl chloroformate in the
presence of a base such as e.g. triethylamine, removal of the
formed triethylammonium chloride by filtration and addition of
lithium borohydride to yield tert-butyl
N-(3-(hydroxylmethyl)benzyl)carbamate. tert-Butyl
N-(3-(hydroxylmethyl)benzyl)carbamate may be oxidized with a
suitable reagent or combination of reagents, e.g. using a Swern
oxidation, known to a person trained in the art, comprising the
addition of a solution of the alcohol in e.g. dichloromethane at
-78.degree. C. to a mixture of oxalyl chloride and
dimethylsulfoxidein dichloromethane, which has been formed at
-78.degree. C., followed by addition of a suitable amino-base such
as e.g. triethylamine and subsequent warming to room temperature.
The formed tert-butyl N-(3-formylbenzyl)carbamate may be reacted to
yield tert-butyl N-(3-((hydroxylimino)methyl)benzyl) by reaction
with the free base or a suitable salt of hydroxylamine in a
solution of e.g. sodium hydroxide in water. The BOC-protection
group may be removed from tert-butyl
N-(3-((hydroxylimino)methyl)benzyl) by methods described in the
literature (e.g. T. W. Green, P. G. M Wuts Protective groups in
organic synthesis 2.sup.nd ed. Wiley, New York, 1991) e.g. by
treatment with a 50% solution of trifluoroacetic acid in
dichloromethane to give 3-(aminomethyl)benzaldehyde oxime. Finally,
4-(mPEG20000yl)-N-(3-(hydroxyimino)benzyl)butanoic amide may be
prepared by amide-forming reaction, comprising a reaction of the
free base or a suitable salt of 3-(aminomethyl)benzaldehyde oxime
in the presence of an excess of a suitable base such as e.g.
ethyidiisopropylamine with commercially available
2,5-dioxypyrrolidinyl 4-(mPEG20000yl)butanoic ester (Nektar,
2M450P01). ##STR17##
[0154] In an alternative second step, a mixture of an appropriate
amount of copper sulphate penthydrate, e.g. 5% or 10% or 1
equivalent or 10 equivalents with respect to
(S)2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide and
an appropriate amount of L-ascorbic acid, such as e.g. 50 eq with
respect to
(S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide,
may be prepared in water, which is buffered with 2,6-lutidine.
After a appropriate period of time such as e.g 5 min, this solution
may be given to a solution of
(S)-2-((hIL-21yl)amino)-3-(4-(proparyloxy)phenyl)propanoic amide
and N-(2-(mPEG20000yl)ethyl) 11-azidoundecanoic amide which is
buffered with 2,6-lutidine. The reaction mixture may be kept at a
appropriate temperature such as e.g. room temperature until a
mixture of a single compound selected from
(S)-((hIL-21)amino)-3-(4-((1-(10-(N-(2-(mPEG20000yl)ethyl)carbamoyl)decan-
yl)-1,2,3-triazol-4-yl)methoxy)phenyl) and (S)-((hIL-21
)amino)-3-(4-((1-(10-(N-(2-(mPEG20000yl)ethyl)carbamoyl)decanyl)-1,2,3-tr-
iazol-5-yl)methoxy)phenyl) and may be formed. ##STR18##
[0155] The synthesis of N-(2-(mPEG20000yl)ethyl)11-azidoundecanoic
amide may be performed by reaction of commercially available methyl
11-bromoundecanoic ester with sodium azide in an appropriate
solvent such as e.g. N,N-dimethylformamide at an appropriate
temperature as e.g. 60.degree. C. The formed methyl
11-azidoundecanoic ester may be saponified by a method known to a
person skilled in the art and described in the literature (e.g. T.
W. Green, P. G. M Wuts Protective groups in organic synthesis
2.sup.nd ed. Wiley, New York, 1991) such as e.g. potassium
hydroxide in methanol or potassium triethylsilanolate in
tetrahydrofuran. The resulting acid may be activated by a method
known to a person skilled in the art e.g. by reaction with
2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU)
in an appropriate solvent such as e.g. N,N-dimethylformamide at an
appropriate temperature such as e.g. room temperature to give
11-azido-N-2,5-dioxopyrrolidin-1-ylundecanoic amide.
11-Azido-N-2,5-dioxopyrrolidin-1-ylundecanoic amide may be reacted
with commercially available (2-(mPEG20000yl)ethyl)amine (Nektar
2M2U0P01) in an appropriate solvent such as e.g. dichloromethane
and in the presence of an appropriate base such as e.g.
triethylamine or ethyldiisopropylamine go give
N-(2-(mPEG20000yl)ethyl)11-azidoundecanoic amide. ##STR19##
EXAMPLE
1-(((((4-((4-(mPEG-20000yl)butanoyl)amino)butoximinoacetyl)serinyl)glutamy-
l)alaninyl)glutamyl)hIL-21
[0156] Step 1:
2-(4-(tert-Butoxycarbonylaminoxy)butyl)isoindole-1,3-dione
[0157] ##STR20##
[0158] To a mixture of commercially available
N-(4-bromobutyl)phthalimide (2.82 g, 10 mmol) and
N-Boc-hydroxylamine (2.08 g, 15.6 mmol) was added acetonitrile (2
ml) and successively 1,8-diazabicyclo[5.4.0]undec-7-ene (2.25 ml,
15 mmol). The reaction mixture was stirred at room temperature for
30 min and then at 50.degree. C. for 2 days. It was diluted with a
mixture of water (30 ml) and 1 N hydrochloric acid (20 ml). It was
extracted with ethyl acetate (2.times.100 ml). The organic phase
was washed with brine (50 ml) and was dried over magnesium
sulphate. The crude product was purified by chromatography on
silica (60 g), using a gradient of heptane/ethyl acetate 1:0 to 0:1
as eluent to give 2.08 g of
2-(4-(tert-butoxycarbonylaminoxy)butyl)isoindole-1,3-dione.
[0159] Step 2:
N-(4-aminobutoxy)carbamic acid tert-butyl ester
[0160] ##STR21##
[0161] Hydrazine hydrate (1.0 ml, 20 mmol) was added to a solution
of 2-(4-(tert-butoxycarbonylaminoxy)butyl)isoindole-1,3-dione (2.08
g, 6.22 mmol) in ethanol (8.0 ml). The reaction mixture was stirred
at 80.degree. C. for 65 h. The solvent was removed in vacuo. The
residue was dissolved in toluene (10 ml) and the solvent was
removed in vacuo. The residue was suspended in 1 N hydrochloric
acid (10 ml). The precipitation was removed by filtration and was
washed with water (2 ml). The filtrate and the wash-liquids were
combined and made basic with potassium carbonate. The solution was
extracted with dichloromethane (4.times.20 ml). The organic layer
was dried over magnesium sulphate. The solvent was removed in vacuo
to give 0.39 g of N-(4-aminobutoxy)carbamic acid tert-butyl ester.
Potassium carbonate (3 g) was added to the aqueous phase, which was
extracted with dichloromethane (3.times.20 ml). These combined
organic layers were dried over magnesium sulphate. The solvent was
removed in vacuo to give another 0.39 g of
N-(4-aminobutoxy)carbamic acid tert-butyl ester.
[0162] Step 3:
N-(4-(4-(mPEG20000yl)butanolyamino)butoxy)carbamic acid tert-butyl
ester
[0163] ##STR22##
[0164] The commercially available N-hydroxysuccinimide ester of
mPEG2000ylbutanoic acid (Nektar "mPEG-SBA", #2M450P01, 3 g, 0.15
mmol) was dissolved in dichloromethane (25 ml).
N-(4-Aminobutoxy)carbamic acid tert-butyl ester (0.12 g, 0.59 mmol)
was added. The reaction mixture was shaken at room temperature.
Diethyl ether was added until a precipitation was obtained. The
precipitation was isolated by filtration. The material was dried in
vacuo to yield 2.39 g of
N-(4-(4-(mPEG20000yl)butanolyamino)butoxy)carbamic acid tert-butyl
ester.
[0165] Step 4:
N-(4-Aminoxybutyl)-4-(mPEG20000yl)butanolyamide
[0166] ##STR23##
[0167] Trifluoroacetic acid (20 ml) was added to a solution of
N-(4-(4-(mPEG20000yl)butanolyamino)butoxy)carbamic acid tert-butyl
ester (2.39 g, 0.12 mmol) in dichloromethane (20 ml). The reaction
mixture was shaken for 30 min. Diethyl ether (100 ml) was added.
The formed precipitation was isolated by filtration. It was washed
with diethyl ether (2.times.100 ml) and dried in vacuo to give 1.96
g of N-(4-aminoxybutyl)-4-(mPEG20000yl)butanolyamide
[0168] Step 5:
[0169] 1-((((Serinyl)glutamyl)alninyl)glutamyl)hIL-21 (4 mg,
lyophilized in a phosphate-buffer, 252 nmol) was dissolved in 0.400
ml of a buffer, consisting of triethanolamine (0.008 ml) in water
(4 ml). A solution of methionine (3.15 mg, 21420 nmol) in water
(0.12 ml) and a solution of sodium periodate (0.38 mg, 1890 nmol)
were added successively. The reaction mixture was left for 30 min
at room temperature. A solution of
N-(4-aminoxybutyl)-4-(mPEG20000yl)butanolyamide (77 mg, 3780 nmol)
in water (0.240 ml) was added. The pH was adjusted to pH 4-5 with
glacial acetic acid (0.004 ml). The reaction mixture was left at
room temperature for 16 h. The reaction mixture was diluted with a
solution of triethanolamine (12 mg) in water (3.2 ml) and was kept
at -18.degree. C. until purification.
[0170] Protein Chemistry
[0171] Recombinant Interleukin 21 (IL21) was expressed as inclusion
bodies in E.coli with a N-terminal extension
(Met-Ser-Glu-Ala-Glu-hIL21). The N-terminal Met residue is removed
by the protease systems present in E.coli, leaving
Ser-Glu-Ala-Glu-hIL21. The Glu-Ala-Glu amino acid sequence can be
present or absent (we initiate experiments with Met-Ser-hIL21).
[0172] The protein was refolded and purified to 90-95% purity using
conventional chromatographic methods.
[0173] The pure protein was subsequently N-terminally pegylated via
reaction described in steps 1-5.
[0174] Subsequent purification was done using gelfiltration or size
exclusion chromatography (SEC).
[0175] In an proliferation assay, the pegylated IL21 showed similar
potentency to the unpegylated standard, indicating that the
pegylation does not interfere with receptor binding, and that the
reaction procedures are not harmful to the protein.
Pharmacological Methods
[0176] Proliferation assay using Baf-3(IL21R) cells.
[0177] IL3 dependent Baf-3 cells transfected with either the murine
or the humane IL21 R are grown in IL-3 containing culture medium
until setup of a proliferation assay (preferably 3 days).
[0178] Cells used for the assay are washed in IL3-free medium and
plated in 96 well-plates with 50,000 c/w in assay media (without
IL-3). IL21 is added in serial dilutions from
10.sup.-7M-10.sup.-13M and the cells incubated at 37.degree. C., 5%
CO.sub.2. AlamarBlue (Biosource) is added to all wells after 66
hours of culture and the cells incubated further for 6 hours. If
cells are growing, the alamarBlue is reduced and the colour of the
media changes from blue to red. Plates are then read on a Fluostar
(bmg) at 550 nm (excitation) and 590 nm (emission) and analysed by
Prism (GrafPad software).
[0179] A ref to Baf-3 cells:
[0180] Palacios, R. & Steinmetz, M. (1985) Cell 41 pp
727-734.
Description of a PEG-hIL-21 PK Study in Mice
[0181] The present experiment is to administer a single dose of
PEG20K-hIL-21, PEG40K-hIL-21 and hIL-21 intravenously and
subcutaneously to mice in order to obtain bioavailability and
pharmacokinetics characteristics of PEG-hIL-21.
Material and Methods
[0182] Forty eight female C57BL/6Jbom weighing approximately 25 g
from Bomholtgard, Ry, Denmark are included in the experiment.
[0183] During the study the animals will be kept and handled
according to normal procedure in the animal unit (Standard
Operating Procedure no. 010364) and are allowed free access to feed
and water.
Test Formulations
[0184] hIL-21, PEG20k-hIL-21 and PEG40k-hIL-21 at a concentration
of 200 .mu.g/ml. The test substances will be dissolved in PBS
buffer pH 7.4.
Dosing
[0185] The test substance will be dosed according to the
following:
[0186] 20 .mu.g/25 g mouse corresponding to 0.8 .mu.g/g mouse
weight.
[0187] The i.v. injections will be given in the tail vein in a
volume of 0.1 ml.
[0188] The s.c. injections will be given on the back of neck in a
volume of 0.1 ml.
Blood Samples
[0189] Blood samples will be collected according to the following
schedule:
After Intravenous Injection:
[0190] Predose, 5, 10, 20, 30, 45 (minutes), 1, 1.5, 2, 4 and 6
hours after dosing.
After Subcutaneous Injection:
[0191] Predose, 10, 30 (minutes), 1, 1.5, 2, 3, 4, 6, 8 and 24
hours after dosing.
[0192] Blood samples will be drawn from the orbital venous plexus.
Approximately 0.1-0.2 ml blood will be drawn at each sampling time.
Three blood samples will be taken from each animal. Blood samples
from two mice will be drawn at each time point.
[0193] Blood samples will be collected in Micronic test tubes and
kept on ice for max 20 min before centrifugation (1200 m.times.g,
4.degree. C., 10 min).
[0194] 25 .mu.l plasma sample will be transferred to Micronic tubes
immediately after centrifugation and stored at -20.degree. C. until
analysis.
[0195] Assay
[0196] The plasma samples will be analysed for the content of
hIL-21 by a specific immunoassay by Immunochemistry, Novo Nordisk
A/S.
[0197] Plasma concentration-time profiles will be analysed by
noncompartmental and compartmental pharmacokinetic methods.
[0198] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law), regardless of any separately provided
incorporation of particular documents made elsewhere herein.
[0199] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context.
[0200] Unless otherwise stated, all exact values provided herein
are representative of corresponding approximate values (e.g., all
exact exemplary values provided with respect to a particular factor
or measurement can be considered to also provide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0201] The description herein of any aspect or embodiment of the
invention using terms such as "comprising", "having," "including,"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or embodiment of
the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
composition described herein as comprising a particular element
should be understood as also describing a composition consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0202] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way.
[0203] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0204] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability, and/or enforceability of such patent
documents.
[0205] This invention includes all modifications and equivalents of
the subject matter recited in the claims and/or aspects appended
hereto as permitted by applicable law.
* * * * *