U.S. patent application number 11/553389 was filed with the patent office on 2007-03-15 for methods of treating infections using il-21.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Wayne Kindsvogel, Andrew J. Nelson.
Application Number | 20070059284 11/553389 |
Document ID | / |
Family ID | 29736266 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070059284 |
Kind Code |
A1 |
Nelson; Andrew J. ; et
al. |
March 15, 2007 |
METHODS OF TREATING INFECTIONS USING IL-21
Abstract
Methods for treating mammals with infections, particularly viral
infections using molecules that have an IL-21 functional activity
are described. The molecules having IL-21 functional activities
include polypeptides that have homology to the human IL-21
polypeptide sequence and proteins fused to a polypeptide with IL-21
functional activity. The molecules can be used as a monotherapy or
in combination with other known antimicrobial or antiviral
therapeutics.
Inventors: |
Nelson; Andrew J.;
(Shoreline, WA) ; Kindsvogel; Wayne; (Seattle,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
29736266 |
Appl. No.: |
11/553389 |
Filed: |
October 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10456262 |
Jun 6, 2003 |
|
|
|
11553389 |
Oct 26, 2006 |
|
|
|
60387127 |
Jun 7, 2002 |
|
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
37/04 20180101; A61K 38/2026 20130101; A61P 31/18 20180101; A61P
37/02 20180101; Y02A 50/475 20180101; A61K 38/20 20130101; A61P
31/10 20180101; A61P 31/12 20180101; A61P 31/16 20180101; A61P
35/04 20180101; A61P 33/02 20180101; Y02A 50/463 20180101; A61P
31/20 20180101; A61P 31/22 20180101; C07K 2317/73 20130101; Y02A
50/465 20180101; A61P 1/04 20180101; A61P 25/00 20180101; A61P
35/00 20180101; A61K 38/2013 20130101; C07K 14/54 20130101; A61P
31/14 20180101; Y02A 50/481 20180101; A61K 38/2086 20130101; C07K
16/2878 20130101; A61K 45/06 20130101; A61K 38/193 20130101; A61P
1/16 20180101; A61P 31/04 20180101; A61P 31/06 20180101; A61P 1/00
20180101; A61P 19/02 20180101; A61P 31/00 20180101; A61P 11/00
20180101; A61P 35/02 20180101 |
Class at
Publication: |
424/085.2 |
International
Class: |
A61K 38/20 20060101
A61K038/20 |
Claims
1. A method of treating an infection comprising administering a
therapeutically effective amount of a polypeptide that has at least
90% identity to an IL-21 polypeptide comprising residues 32 (Gln)
to 162 (Ser) of SEQ ID NO: 2, wherein the infection are Influenza
viruses;.
2. The method according to claim 1, wherein the polypeptide has at
least 95% identity to an IL-21 polypeptide comprising residues 32
(Gln) to 162 (Ser) of SEQ ID NO: 2.
3. The method according claim 1, wherein the polypeptide is an
IL-21 polypeptide comprising residues 32 (Gln) to 162 (Ser) of SEQ
ID NO: 2.
4. A method of treating an infection comprising administering a
therapeutically effective amount of a fusion protein comprising a
first polypeptide that has at least 90% identity to an IL-21
polypeptide comprising residues 32 (Gln) to 162 (Ser) of SEQ ID NO:
2, and a second polypeptide, wherein the infection are Influenza
viruses.
5. The method according to claim 1 such that the level of viral
infection is reduced.
6. The method according to claim 1 wherein a reduction in the level
of viral infection is measured as reduction in viral load,
increased viral-specific antibodies, reduction in alanine
aminotransferase level (ALT), or histologic improvement in a target
tissue as measured by immunohistochemistry.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/456,262, filed Jun. 6, 2003, which is a
continuation-in-part which claims benefit of U.S. Provisional
Application Ser. No. 60/387,127, filed on Jun. 7, 2002, and is
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Cytokines generally stimulate proliferation or
differentiation of cells of the hematopoietic lineage or
participate in the immune and inflammatory response mechanisms of
the body. Examples of cytokines which affect hematopoiesis are
erythropoietin (EPO), which stimulates the development of red blood
cells; thrombopoietin (TPO), which stimulates development of cells
of the megakaryocyte lineage; and granulocyte-colony stimulating
factor (G-CSF), which stimulates development of neutrophils. These
cytokines are useful in restoring normal blood cell levels in
patients suffering from anemia, thrombocytopenia, and neutropenia
or receiving chemotherapy for cancer.
[0003] The interleukins are a family of cytokines that mediate
immunological responses. Central to an immune response is the T
cell, which produce many cytokines and adaptive immunity to
antigens. Cytokines produced by the T cell have been classified as
type 1 and type 2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998).
Type 1 cytokines include IL-2, IFN-.gamma., LT-.alpha., and are
involved in inflammatory responses, viral immunity, intracellular
parasite immunity and allograft rejection. Type 2 cytokines include
IL-4, IL-5, IL-6, IL-10 and IL-13, and are involved in humoral
responses, helminth immunity and allergic response. Shared
cytokines between Type 1 and 2 include IL-3, GM-CSF and
TNF-.alpha.. There is some evidence to suggest that Type 1 and Type
2 producing T cell populations preferentially migrate into
different types of inflamed tissue.
[0004] Mature T cells can activated, i.e., by an antigen or other
stimulus, to produce, for example, cytokines, biochemical signaling
molecules, or receptors that further influence the fate of the T
cell population.
[0005] B cells can be activated via receptors on their cell surface
including B cell receptor and other accessory molecules to perform
accessory cell functions, such as production of cytokines.
[0006] Natural killer (NK) cells have a common progenitor cell with
T cells and B cells, and play a role in immune surveillance. NK
cells, which comprise up to 15% of blood lymphocytes, do not
express antigen receptors, and therefore do not use MHC recognition
as requirement for binding to a target cell. NK cells are involved
in the recognition and killing of certain tumor cells and virally
infected cells. In vivo, NK cells are believed to require
activation, however, in vitro, NK cells have been shown to kill
some types of tumor cells without activation.
[0007] Lymphomas are malignancies of the lymphatic system, that are
heterogenous in etiology, morphology, and clinical course.
Lymphomas are generally classified as either Hodgkins disease or
Non-Hodgkins lymphomas. Hodgkins disease is characterized by giant
histocytes, whereas absence of the cells encompasses all
non-Hodgkins lymphomas. Lymphocytes, which are the primary
component of lymph, can be B cell lymphocytes or T cell
lymphocytes. Generally, when a lymphoma arises early in cell
maturation, the malignancy is more aggressive than malignancies
arising from mature cells. Chemotherapy is usually more effective
in treating aggressive lymphoma, whereas indolent lymphomas cannot
be treated as easily and therefore are likely never to be cured as
long as the disease remains indolent. For a survey of information
relating to lymphoma, see, e.g. Lymphoma Treatments and Managing
Their Side Effects, Lymphoma Res. Found. Of Amer., Los Angeles,
2001.
[0008] In other aspects, the present invention provides such
methods for treating solid tumors and lymphomas by administrating
IL-21 compositions that may used as a monotherapy or in combination
with chemotherapy, radiation therapy, or other biologics. These and
other uses should be apparent to those skilled in the art from the
teachings herein.
SUMMARY OF THE INVENTION
[0009] Within one aspect, the present invention provides a method
of treating Non-Hodgskins lymphoma comprising administering to a
subject in need thereof a therapeutically effective amount of a
polypeptide having a functional activity of IL-21. In certain
embodiments, the polypeptide has been shown to not cause
proliferation of isolated cancer cells prior to administration to
the subject.
[0010] In another aspect, the present invention provides a method
of treating cancer comprising administering to subject a
therapeutically effective amount of a polypeptide having a
functional activity of IL-21, wherein the cancer is selected from
the group of renal cell carcinoma, epithelial carcinoma, breast
cancer, prostate cancer, ovarian cancer and colon cancer. In one
embodiment, there is a tumor response. In another embodiment, the
tumor response is measured as complete response, partial response
or reduction in time to progression.
[0011] In another aspect, the present invention provides a method
of treating Non-Hodgskins lymphoma comprising administering to a
subject in need thereof a therapeutically effective amount of a
fusion protein comprising a first polypeptide having a functional
activity of IL-21 and a second polypeptide. In other embodiments,
the methods provide the cancer is selected from the group of renal
cell carcinoma, epithelial carcinoma, breast cancer, prostate
cancer, ovarian cancer and colon cancer. In one embodiment, there
is a tumor response. In another embodiment, the tumor response is
measured as complete response, partial response or reduction in
time to progression.
DESCRIPTION OF THE INVENTION
[0012] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0013] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzmol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, 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.).
[0014] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0015] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0016] The term "cancer" or "cancer cell" is used herein to denote
a tissue or cell found in a neoplasm which possesses
characteristics which differentiate it from normal tissue or tissue
cells. Among such characteristics include but are not limited to:
degree of anaplasia, irregularity in shape, indistinctness of cell
outline, nuclear size, changes in structure of nucleus or
cytoplasm, other phenotypic changes, presence of cellular proteins
indicative of a cancerous or pre-cancerous state, increased number
of mitoses, and ability to metastasize. Words pertaining to
"cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia,
lymphoma, polyp, and scirrus, transformation, neoplasm, and the
like.
[0017] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0018] The term "complements of a polynucleotide molecule" denotes
a polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence.
[0019] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0020] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0021] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0022] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0023] The term "level" when referring to immune cells, such as NK
cells, T cells, in particular cytotoxic T cells, B cells and the
like, an increased level is either increased number of cells or
enhanced activity of cell function.
[0024] The term "level" when referring to viral infections refers
to a change in the level of viral infection and includes, but is
not limited to, a change in the level of CTLs or NK cells (as
described above), a decrease in viral load, an increase antiviral
antibody titer, decrease in serological levels of alanine
aminotransferase, or improvement as determined by histological
examination of a target tissue or organ. Determination of whether
these changes in level are significant differences or changes is
well within the skill of one in the art.
[0025] The term "neoplastic", when referring to cells, indicates
cells undergoing new and abnormal proliferation, particularly in a
tissue where in the proliferation is uncontrolled and progressive,
resulting in a neoplasm. The neoplastic cells can be either
malignant, i.e. invasive and metastatic, or benign.
[0026] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0027] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0028] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0029] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0030] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0031] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0032] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0033] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0034] All references cited herein are incorporated by reference in
their entirety.
[0035] The present invention is based in part upon the discovery
that administration of IL-21 results in inhibiting proliferation of
certain neoplastic cells, either directly or indirectly, thereby
limiting the pathological effects caused by specific cancers. In
the examples which follow, animal models and in vitro assays
demonstrate the activity of IL-21 on biological samples.
A. Description of IL-21 and its Receptor.
[0036] Human IL-21 (SEQ ID NO:1 and SEQ ID NO:2) was designated
IL-21, and is described in commonly-owned U.S. Pat. No. 6,307,024,
which is incorporated herein by reference. The IL-21 receptor,
(previously designated zalphal I) now designated IL-21R (SEQ ID
NO:5 and SEQ ID NO:6), and heterodimeric receptor
IL-21R/IL-2R.gamma. are described in commonly-owned WIPO
Publication Nos. WO 0/17235 and WO 01/77171, which are incorporated
herein by reference. As described in these publications, IL-21 was
isolated from a cDNA library generated from activated human
peripheral blood cells (hPBCs), which were selected for CD3. CD3 is
a cell surface marker unique to cells of lymphoid origin,
particularly T cells.
[0037] The amino acid sequence for the IL-21R indicated that the
encoded receptor belonged to the Class I cytokine receptor
subfamily that includes, but is not limited to, the receptors for
IL-2, IL-4, IL-7, IL-15, EPO, TPO, GM-CSF and G-CSF (for a review
see, Cosman, "The Hematopoietin Receptor Superfamily" in Cytokine
5(2): 95-106, 1993). The tissue distribution of the receptor
suggests that a target for IL-21 is hematopoietic lineage cells, in
particular lymphoid progenitor cells and lymphoid cells. Other
known four-helical-bundle cytokines that act on lymphoid cells
include IL-2, IL-4, IL-7, and IL- 15. For a review of
four-helical-bundle cytokines, see, Nicola et al., Advances in
Protein Chemistry 52:1-65, 1999 and Kelso, A., Immunol. Cell Biol.
76:300-317, 1998.
[0038] For IL-21, the secretory signal sequence is comprised of
amino acid residues 1 (Met) to 31 (Gly), and the mature polypeptide
is comprised of amino acid residues 32 (Gln) to 162 (Ser) (as shown
in SEQ ID NO: 2). In general, cytokines are predicted to have a
four-alpha helix structure, with helices A, C and D being most
important in ligand-receptor interactions, and are more highly
conserved among members of the family. Referring to the human IL-21
amino acid sequence shown in SEQ ID NO:2, an alignment of human
IL-21, human IL-15, human IL-4, and human GM-CSF amino acid
sequences predicted that IL-21 helix A is defined by amino acid
residues 41-56; helix B by amino acid residues 69-84; helix C by
amino acid residues 92-105; and helix D by amino acid residues
135-148; as shown in SEQ ID NO: 2. Structural analysis suggests
that the A/B loop is long, the B/C loop is short and the C/D loop
is parallel long. This loop structure results in an up-up-down-down
helical organization. The cysteine residues are absolutely
conserved between IL-21 and IL-15. The cysteine residues that are
conserved between IL-15 and IL-21 correspond to amino acid residues
71, 78, 122 and 125 of SEQ ID NO: 2. Conservation of some of the
cysteine residues is also found in IL-2, IL-4, GM-CSF and IL-21
corresponding to amino acid residues 78 and 125 of SEQ ID NO: 2.
Consistent cysteine placement is further confirmation of the
four-helical-bundle structure. Also highly conserved in the family
comprising IL-15, IL-2, IL-4, GM-CSF and IL-21 is the Glu-Phe-Leu
sequence as shown in SEQ ID NO: 2 at residues 136-138. Further
analysis of IL-21 based on multiple alignments predicts that amino
acid residues 44, 47 and 135 (as shown in SEQ ID NO: 2) play an
important role in IL-21 binding to its cognate receptor. Moreover,
the predicted amino acid sequence of murine IL-21 (SEQ ID NO:4)
shows 57% identity to the predicted human protein. Based on
comparison between sequences of human and murine IL-21
well-conserved residues were found in the regions predicted to
encode alpha helices A and D.
[0039] The corresponding polynucleotides encoding the IL-21
polypeptide regions, domains, motifs, residues and sequences
described herein are as shown in SEQ ID NO:1. The amino acid
residues comprising helices A, B, C, and D, and loops A/B, B/C and
C/D for IL-21 , IL-2, IL-4, IL-15 and GM-CSF are shown in Table 1.
TABLE-US-00001 TABLE 1 A/B B/C C/D Helix A Loop Helix B Loop Helix
C Loop Helix D IL- 41-56 57-68 69-84 85-91 92-105 106-134 135-148
SEQ ID 21residues NO: 2 IL-2 36-46 47-52 53-75 76-86 87-99 100-102
103-121 SEQ ID residues NO: 5 IL-4 29-43 44-64 65-83 84-94 95-118
119-133 134-151 SEQ ID residues NO: 6 IL-15 45-68 69-83 84-101
102-106 107-119 120-133 134-160 SEQ ID residues NO: 7 GM-CSF 30-44
45-71 72-81 82-90 91-102 103-119 120-131 SEQ ID residues NO: 8
[0040] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human IL-21
and that allelic variation and alternative splicing are expected to
occur. Allelic variants of this sequence can be cloned by probing
cDNA or genomic libraries from different individuals according to
standard procedures. Allelic variants of the DNA sequence shown in
SEQ ID NO:1, including those containing silent mutations and those
in which mutations result in amino acid sequence changes, are
within the scope of the present invention, as are proteins which
are allelic variants of SEQ ID NO:2. cDNAs generated from
alternatively spliced mRNAs, which retain the properties of the
IL-21 polypeptide, are included within the scope of the present
invention, as are polypeptides encoded by such cDNAs and mRNAs.
Allelic variants and splice variants of these sequences can be
cloned by probing cDNA or genomic libraries from different
individuals or tissues according to standard procedures known in
the art.
[0041] The present invention also provides isolated IL-21
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides comprising at least 70%, at least 80%, at least 90%,
at least 95%, or greater than 95% sequence identity to the
sequences shown in SEQ ID NO:2, or their orthologs. The present
invention also includes polypeptides that comprise an amino acid
sequence having at least 70%, at least 80%, at least 90%, at least
95% or greater than 95% sequence identity to the sequence of amino
acid residues 1 to 162 or 33 to 162 of SEQ ID NO:2. The present
invention further includes nucleic acid molecules that encode such
polypeptides. Methods for determining percent identity are
described below.
[0042] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 2 (amino acids are
indicated by the standard one-letter codes). Total .times. .times.
number .times. .times. of .times. .times. identical .times. .times.
matches [ length .times. .times. of .times. .times. the .times.
.times. longer .times. .times. sequence .times. .times. plus
.times. .times. the number .times. .times. of .times. .times. gaps
.times. .times. introduced .times. .times. inot .times. .times. the
.times. .times. longer sequence .times. .times. in .times. .times.
order .times. .times. to .times. .times. align .times. .times. the
.times. .times. two .times. .times. sequences ] .times. 100
##EQU1## TABLE-US-00002 TABLE 2 A R N D C Q E G H I L K M F P S T W
Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5
E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I
-1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1
2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F
-2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2
-3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0
-1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2
-2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2
-1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0
-3 -1 4
[0043] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant IL-21. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
[0044] Variant IL-21 polypeptides or polypeptides with
substantially similar sequence identity are characterized as having
one or more amino acid substitutions, deletions or additions. These
changes are preferably of a minor nature, that is conservative
amino acid substitutions (see Table 3) and other substitutions that
do not significantly affect the folding or activity of the
polypeptide; small deletions, typically of one to about 30 amino
acids; and amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes polypeptides of from about 108 to 216 amino acid
residues that comprise a sequence that is at least 80%, preferably
at least 90%, and more preferably 95%, 96%, 97%, 98%, 99% or more
identical to the corresponding region of SEQ ID NO:2. Polypeptides
comprising affinity tags can further comprise a proteolytic
cleavage site between the IL-21 polypeptide and the affinity tag.
Preferred such sites include thrombin cleavage sites and factor Xa
cleavage sites. TABLE-US-00003 TABLE 3 Conservative amino acid
substitutions Basic: arginine lysine histidine Acidic: glutamic
acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine
isoleucine valine Aromatic: phenylalanine tryptophan tyrosine
Small: glycine alanine serine threonine methionine
[0045] Determination of amino acid residues that comprise regions
or domains that are critical to maintaining structural integrity
can be determined. Within these regions one can determine specific
residues that will be more or less tolerant of change and maintain
the overall tertiary structure of the molecule. Methods for
analyzing sequence structure include, but are not limited to
alignment of multiple sequences with high amino acid or nucleotide
identity, secondary structure propensities, binary patterns,
complementary packing and buried polar interactions (Barton,
Current Opin: Struct. Biol. 5:372-376, 1995 and Cordes et al.,
Current Opin. Struct. Biol. 6:3-10, 1996). In general, when
designing modifications to molecules or identifying specific
fragments determination of structure will be accompanied by
evaluating activity of modified molecules.
[0046] Amino acid sequence changes are made in IL-21 polypeptides
so as to minimize disruption of higher order structure essential to
biological activity. For example, where the IL-21 polypeptide
comprises one or more helices, changes in amino acid residues will
be made so as not to disrupt the helix geometry and other
components of the molecule where changes in conformation abate some
critical function, for example, binding of the molecule to its
binding partners, e.g., A and D helices, residues 44, 47 and 135 of
SEQ ID NO: 2. The effects of amino acid sequence changes can be
predicted by, for example, computer modeling as disclosed above or
determined by analysis of crystal structure (see, e.g., Lapthorn et
al., Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are
well known in the art compare folding of a variant protein to a
standard molecule (e.g., the native protein). For example,
comparison of the cysteine pattern in a variant and standard
molecules can be made. Mass spectrometry and chemical modification
using reduction and alkylation provide methods for determining
cysteine residues which are associated with disulfide bonds or are
free of such associations (Bean et al., Anal. Biochem. 201:216-226,
1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al.,
Anal. Chem. 66:3727-3732, 1994). It is generally believed that if a
modified molecule does not have the same cysteine pattern as the
standard molecule folding would be affected. Another well known and
accepted method for measuring folding is circular dichrosism (CD).
Measuring and comparing the CD spectra generated by a modified
molecule and standard molecule is routine (Johnson, Proteins
7:205-214, 1990). Crystallography is another well known method for
analyzing folding and structure. Nuclear magnetic resonance (NMR),
digestive peptide mapping and epitope mapping are also known
methods for analyzing folding and structurally similarities between
proteins and polypeptides (Schaanan et al., Science 257:961-964,
1992).
[0047] A Hopp/Woods hydrophilicity profile of the IL-21 protein
sequence as shown in SEQ ID NO:2 can be generated (Hopp et al.,
Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth.
88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169,
1998). The profile is based on a sliding six-residue window. Buried
G, S, and T residues and exposed H, Y, and W residues were ignored.
For example, in IL-21, hydrophilic regions include amino acid
residues 114-119 of SEQ ID NO: 2, amino acid residues 101-105 of
SEQ ID NO: 2, amino acid residues 126-131 of SEQ ID NO: 2, amino
acid residues 113-118 of SEQ ID NO: 2, and amino acid residues
158-162 of SEQ ID NO: 2.
[0048] Those skilled in the art will recognize that hydrophilicity
or hydrophobicity will be taken into account when designing
modifications in the amino acid sequence of a IL-21 polypeptide, so
as not to disrupt the overall structural and biological profile. Of
particular interest for replacement are hydrophobic residues
selected from the group consisting of Val, Leu and Ile or the group
consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example,
residues tolerant of substitution could include residues 100 and
103 as shown in SEQ ID NO: 2. Cysteine residues at positions 71,
78, 122 and 125 of SEQ ID NO: 2, will be relatively intolerant of
substitution.
[0049] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between IL-15, IL-2, IL-4 and
GM-CSF with IL-21. Using methods such as "FASTA" analysis described
previously, regions of high similarity are identified within a
family of proteins and used to analyze amino acid sequence for
conserved regions. An alternative approach to identifying a variant
IL-21 polynucleotide on the basis of structure is to determine
whether a nucleic acid molecule encoding a potential variant IL-21
gene can hybridize to a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1, as discussed above.
[0050] Other methods of identifying essential amino acids in the
polypeptides of the present invention are procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological or biochemical
activity as disclosed below to identify amino acid residues that
are critical to the activity of the molecule. See also, Hilton et
al., J. Biol. Chem. 271:4699 (1996).
[0051] The present invention also includes administration of
molecules having the functional activity of IL-21. Thus,
administration of functional fragments and functional modified
polypeptides of IL-21 polypeptides and nucleic acid molecules
encoding such functional fragments and modified polypeptides. A
"functional" IL-21 or fragment thereof as defined herein is
characterized by its proliferative or differentiating activity, by
its ability to induce or inhibit specialized cell functions, in
particular for immune effector cells, such as NK cells, T cells, B
cells and dendritic cells. Functional IL-21 also includes the
ability to exhibit anti-cancer and anti-viral effects in vitro or
in vivo, or by its ability to bind specifically to an anti- IL-21
antibody or IL-21 receptor (either soluble or immobilized). As
previously described herein, IL-21 is characterized by a
four-helical-bundle structure comprising helix A (amino acid
residues 41-56), helix B (amino acid residues 69-84), helix C
(amino acid residues 92-105) and helix D (amino acid residues
135-148), as shown in SEQ ID NO: 2. Thus, the present invention
further provides fusion proteins encompassing: (a) polypeptide
molecules comprising one or more of the helices described above;
and (b) functional fragments comprising one or more of these
helices. The other polypeptide portion of the fusion protein can
contributed by another four-helical-bundle cytokine, such as IL-15,
IL-2, IL-4 and GM-CSF, or by a non-native and/or an unrelated
secretory signal peptide that facilitates secretion of the fusion
protein.
[0052] Routine deletion analyses of nucleic acid molecules can be
performed to obtain functional fragments of a nucleic acid molecule
that encodes a IL-21 polypeptide. As an illustration, DNA molecules
having the nucleotide sequence of SEQ ID NO:1 or fragments thereof,
can be digested with Bal31 nuclease to obtain a series of nested
deletions. These DNA fragments are then inserted into expression
vectors in proper reading frame, and the expressed polypeptides are
isolated and tested for IL-21 activity, or for the ability to bind
anti-IL-21 antibodies or zalphal 1 receptor. One alternative to
exonuclease digestion is to use oligonucleotide-directed
mutagenesis to introduce deletions or stop codons to specify
production of a desired IL-21 fragment. Alternatively, particular
fragments of a IL-21 gene can be synthesized using the polymerase
chain reaction.
[0053] Standard methods for identifying functional domains are
well-known to those of skill in the art. For example, studies on
the truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993); Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation 1 Boynton et al., (eds.) pages 169-199 (Academic
Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270 (1995);
Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al.,
Biochem. Pharmacol. 50:1295 (1995); and Meisel et al., Plant Molec.
Biol. 30:1 (1996).
[0054] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204), and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)).
[0055] Variants of the disclosed IL-21 nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Natl Acad. Sci.
USA 91:10747 (1994), and international publication No. WO 97/20078.
Briefly, variant DNA molecules are generated by in vitro homologous
recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point
mutations. This technique can be modified by using a family of
parent DNA molecules, such as allelic variants or DNA molecules
from different species, to introduce additional variability into
the process. Selection or screening for the desired activity,
followed by additional iterations of mutagenesis and assay provides
for rapid "evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against detrimental
changes.
[0056] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-IL-21 antibodies or soluble
zalphal 1 receptor, can be recovered from the host cells and
rapidly sequenced using modern equipment. These methods allow the
rapid determination of the importance of individual amino acid
residues in a polypeptide of interest, and can be applied to
polypeptides of unknown structure.
[0057] In addition, the proteins of the present invention (or
polypeptide fragments thereof) can be joined to other bioactive
molecules, particularly other cytokines, to provide
multi-functional molecules. For example, one or more helices from
IL-21 can be joined to other cytokines to enhance their biological
properties or efficiency of production.
[0058] The present invention thus provides a series of novel,
hybrid molecules in which a segment comprising one or more of the
helices of IL-21 is fused to another polypeptide. Fusion is
preferably done by splicing at the DNA level to allow expression of
chimeric molecules in recombinant production systems. The resultant
molecules are then assayed for such properties as improved
solubility, improved stability, prolonged clearance half-life,
improved expression and secretion levels, and pharmacodynamics.
Such hybrid molecules may further comprise additional amino acid
residues (e.g. a polypeptide linker) between the component proteins
or polypeptides.
[0059] Non-naturally occurring amino acids include, without
limitation, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,
allo-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 non-naturally 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. Transcription and
translation of plasmids containing nonsense mutations is typically
carried out in a cell-free system comprising an E. coli S30 extract
and commercially available enzymes and other reagents. Proteins are
purified by chromatography. See, for example, Robertson et al., J.
Am. Chem. Soc. 113:2722 (1991), Ellman et al., Methods Enzymol.
202:301 (1991), Chung et al., Science 259:806 (1993), and Chung et
al., Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0060] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcaffi et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993). It can
advantageous to stabilize IL-21 to extend the half-life of the
molecule, particularly for extending metabolic persistence in an
active state. To achieve extended half-life, IL-21 molecules can be
chemically modified using methods described herein. PEGylation is
one method commonly used that has been demonstrated to increase
plasma half-life, increased solubility, and decreased antigenicity
and immunogenicity (Nucci et al., Advanced Drum Delivery Reviews
6:133-155, 1991 and Lu et al., Int. J. Peptide Protein Res.
43:127-138, 1994).
[0061] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids can substituted
for IL-21 amino acid residues.
[0062] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a IL-21
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0063] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein. Hopp/Woods hydrophilicity
profiles can be used to determine regions that have the most
antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,
ibid.). In IL-21 these regions include: amino acid residues
114-119, 101-105, 126-131, 113-118, and 158-162 of SEQ ID NO:
2.
[0064] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fourteen amino acids, or about fourteen to about thirty amino
acids of SEQ ID NO:2 or SEQ ID NO:4. Such epitope-bearing peptides
and polypeptides can be produced by fragmenting a IL-21
polypeptide, or by chemical peptide synthesis, as described herein.
Moreover, epitopes can be selected by phage display of random
peptide libraries (see, for example, Lane and Stephen, Curr. Opin.
Immunol. 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.
7:616 (1996)). Standard methods for identifying epitopes and
producing antibodies from small peptides that comprise an epitope
are described, for example, by Mole, "Epitope Mapping," in Methods
in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The
Humana Press, Inc. 1992); Price, "Production and Characterization
of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0065] Regardless of the particular nucleotide sequence of a
variant IL-21 polynucleotide, the polynucleotide encodes a
polypeptide that is characterized by its proliferative or
differentiating activity, its ability to induce or inhibit
specialized cell functions, or by the ability to bind specifically
to an anti-IL-21 antibody or zalphal 1 receptor. More specifically,
variant IL-21 polynucleotides will encode polypeptides which
exhibit at least 50% and preferably, greater than 70%, 80% or 90%,
of the activity of the polypeptide as shown in SEQ ID NO: 2.
[0066] For any IL-21 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the genetic code and methods known in the art.
[0067] 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. Preferred dimerizing
proteins in this regard include immunoglobulin constant region
domains. Immunoglobulin- IL-21 polypeptide fusions can be expressed
in genetically engineered cells (to produce a variety of multimeric
IL-21 analogs). Auxiliary domains can be fused to IL-21
polypeptides to target them to specific cells, tissues, or
macromolecules. For example, a IL-21 polypeptide or protein could
be targeted to a predetermined cell type by fusing a IL-21
polypeptide to a ligand that specifically binds to a receptor on
the surface of that target cell. In this way, polypeptides and
proteins can be targeted for therapeutic or diagnostic purposes. A
IL-21 polypeptide can be fused to two or more moieties, such as an
affinity tag for purification and a targeting domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly
between domains. See, Tuan et al., Connective Tissue Research
34:1-9, 1996.
[0068] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptides that
have substantially similar sequence identity to residues 1-162 or
33-162 of SEQ ID NO: 2, or functional fragments and fusions
thereof, wherein such polypeptides or fragments or fusions retain
the properties of the wild-type protein such as the ability to
stimulate proliferation, differentiation, induce specialized cell
function or bind the IL-21 receptor or IL-21 antibodies.
[0069] The IL-21 polypeptides used in the present invention 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., eds., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc., NY,
1987.
[0070] In general, a DNA sequence encoding a IL-21 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0071] To direct a 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
IL-21, or may be derived from another secreted protein (e.g., t-PA)
or synthesized de novo. The secretory signal sequence is operably
linked to the IL-21 DNA sequence, i.e., the two sequences are
joined in the correct reading frame and positioned to direct the
newly synthesized polypeptide into the secretory pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to
the DNA sequence encoding the polypeptide of interest, although
certain secretory 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).
[0072] Cultured mammalian cells are suitable hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al.,
ibid.), and liposome-mediated transfection (Hawley-Nelson et al.,
Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral
vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and
Finer, Nature Med. 2:714-6, 1996).
[0073] A wide variety of suitable recombinant host cells includes,
but is not limited to, gram-negative prokaryotic host organisms.
Suitable strains of E. coli include W3110, K12-derived strains
MM294, TG-1, JM-107, BL21, and UT5600. Other suitable strains
include: BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5,
DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101,
JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, ER1647,
E. coli K12, E. coli K12 RV308, E. coli K12 C600, E. coli HB101, E.
coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see, for example,
Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Other
gram-negative prokaryotic hosts can include Serratia, Pseudomonas,
Caulobacter. Prokaryotic hosts can include gram-positive organisms
such as Bacillus, for example, B. subtilis and B. thuringienesis,
and B. thuringienesis var. israelensis, as well as Streptomyces,
for example, S. lividans, S. ambofaciens, S. fradiae, and S.
griseofuscus. Suitable strains of Bacillus subtilus include BR151,
YB886, MI119, MI120, and B170 (see, for example, Hardy, "Bacillus
Cloning Methods," in DNA Cloning: A Practical Approach, Glover
(ed.) (IRL Press 1985)). Standard techniques for propagating
vectors in prokaryotic hosts are well-known to those of skill in
the art (see, for example, Ausubel et al (eds.), Short Protocols in
Molecular Biology, 3.sup.rd Edition (John Wiley & Sons 1995);
Wu et al, Methods in Gene Biotechnology (CRC Press, Inc. 1997)). In
one embodiment, the methods of the present invention use IL-21
expressed in the W3110 strain, which has been deposited at the
American Type Culture Collection (ATCC) as ATCC # 27325.
[0074] When large scale production of IL-21 using the expression
system of the present invention is required, batch fermentation can
be used. Generally, batch fermentation comprises that a first stage
seed flask is prepared by growing E. coli strains expressing IL-21
in a suitable medium in shake flask culture to allow for growth to
an optical density (OD) of between 5 and 20 at 600 nm. A suitable
medium would contain nitrogen from a source(s) such as ammonium
sulfate, ammonium phosphate, ammonium chloride, yeast extract,
hydrolyzed animal proteins, hydrolyzed plant proteins or hydrolyzed
caseins. Phosphate will be supplied from potassium phosphate,
ammonium phosphate, phosphoric acid or sodium phosphate. Other
components would be magnesium chloride or magnesium sulfate,
ferrous sulfate or ferrous chloride, and other trace elements.
Growth medium can be supplemented with carbohydrates, such as
fructose, glucose, galactose, lactose, and glycerol, to improve
growth. Alternatively, a fed batch culture is used to generate a
high yield of IL-21 protein. The IL-21 producing E. coli strains
are grown under conditions similar to those described for the first
stage vessel used to inoculate a batch fermentation.
[0075] Following fermentation the cells are harvested by
centrifugation, re-suspended in homogenization buffer and
homogenized, for example, in an APV-Gaulin homogenizer (Invensys
APV, Tonawanda, N.Y.) or other type of cell disruption equipment,
such as bead mills or sonicators. Alternatively, the cells are
taken directly from the fermentor and homogenized in an APV-Gaulin
homogenizer. The washed inclusion body prep can be solubilized
using guanidine hydrochloride (5-8 M) or urea (7-8 M) containing a
reducing agent such as beta mercaptoethanol (10-100 mM) or
dithiothreitol (5-50 mM). The solutions can be prepared in Tris,
phopshate, HEPES or other appropriate buffers. Inclusion bodies can
also be solubilized with urea (2-4 M) containing sodium lauryl
sulfate (0. 1-2%). In the process for recovering purified IL-21
from transformed E. coli host strains in which the IL-21 is
accumulates as refractile inclusion bodies, the cells are disrupted
and the inclusion bodies are recovered by centrifugation. The
inclusion bodies are then solubilized and denatured in 6 M
guanidine hydrochloride containing a reducing agent. The reduced
IL-21 is then oxidized in a controlled renaturation step. Refolded
IL-21 can be passed through a filter for clarification and removal
of insoluble protein. The solution is then passed through a filter
for clarification and removal of insoluble protein. After the IL-21
protein is refolded and concentrated, the refolded IL-21 protein is
captured in dilute buffer on a cation exchange column and purified
using hydrophobic interaction chromatography.
[0076] It is preferred to purify the polypeptides of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified polypeptide
is substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0077] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which bind to IL-21 proteins or
polypeptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold
Spring Harbor Laboratory Press, 1988. Representative examples of
such assays include: concurrent immunoelectrophoresis,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot assay,
inhibition or competition assay, and sandwich assay. In addition,
antibodies can be screened for binding to wild-type versus mutant
IL-21 protein or polypeptide.
[0078] The methods of the present invention also contemplate using
chemically modified IL-21 compositions, in which a IL-21
polypeptide is linked with a polymer. 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.
[0079] 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. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A IL-21
conjugate can also comprise a mixture of such water-soluble
polymers.
B. The Use of IL-21 for Treating Cancer
[0080] Differentiation is a progressive and dynamic process,
beginning with pluripotent stem cells and ending with terminally
differentiated cells. Pluripotent stem cells that can regenerate
without commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may
or may not continue to be expressed as the cells progress down the
cell lineage pathway toward maturation. Differentiation markers
that are expressed exclusively by mature cells are usually
functional properties such as cell products, enzymes to produce
cell products, and receptors. The stage of a cell population's
differentiation is monitored by identification of markers present
in the cell population.
[0081] There is evidence to suggest that factors that stimulate
specific cell types down a pathway towards terminal differentiation
or dedifferentiation affect the entire cell population originating
from a common precursor or stem cell. Thus, the present invention
includes stimulating or inhibiting the proliferation of lymphoid
cells, hematopoietic cells and epithelial cells.
[0082] IL-21 was isolated from tissue known to have important
immunological function and which contain cells that play a role in
the immune system. IL-21 is expressed in CD3+ selected, activated
peripheral blood cells, and it has been shown that IL-21 expression
increases after T cell activation. Moreover, results of experiments
described in the Examples section herein demonstrate that
polypeptides of the present invention have an effect on the
growth/expansion and/or differentiated state of NK cells or NK
progenitors. Factors that both stimulate proliferation of
hematopoietic progenitors and activate mature cells are generally
known. NK cells are responsive to IL-2 alone, but proliferation and
activation generally require additional growth factors. For
example, it has been shown that IL-7 and Steel Factor (c-kit
ligand) were required for colony formation of NK progenitors.
IL-15+IL-2 in combination with IL-7 and Steel Factor was more
effective (Mrozek et al., Blood 87:2632-2640, 1996). However,
unidentified cytokines may be necessary for proliferation of
specific subsets of NK cells and/or NK progenitors (Robertson et.
al., Blood 76:2451-2438, 1990). A composition comprising IL-21 and
IL-15 stimulates NK progenitors and NK cells, with evidence that
this composition is more potent than previously described factors
and combinations of factors. Moreover, IL-21 promotes NK-cell
expansion, and IL-21 can largely overcome the inhibitory effects of
IL-4 on NK-cell growth, it synergizes with IL-2 to promote NK cell
growth, and IL-21 selectively promotes the expression of
IFN-.gamma. and depresses IL-13 expression. These data suggest that
IL-21 have an indirect role in treating solid tumors, metastatic
tumors and lymphomas by stimulating the immune effector cells
resulting in anti-lymphoma activity. In addition, for certain
cancerous cells where the IL-21 receptor is expressed, the
anticancer effect of IL-21 can be direct.
[0083] Additional evidence demonstrates that IL-21 affects
proliferation and/or differentiation of T cells and B cells in
vivo. It is shown that IL-21 can either inhibit or enhance the
proliferation of normal B cells depending on the nature of the
co-stimulus provided the cells. IL-21 inhibits the proliferation of
some B cell lines, but not others even though most non-responder
cell lines express IL-2IR as measured by specific IL-21 binding.
Many human B cell lines will grow in and kill SCID mice Bonnefoix
et al., Leukemia and Lymphoma 25:169-178, 1997). Examples herein
describe three B-cell lines which are inhibited by IL-21 and three
B cell lines which did not respond to IL-21. All of the cell lines
were IL-21R positive, and were put into SCID mice to determine if
IL-21 could prolong the survival of lymphoma bearing animals. IL-21
exhibited significant efficacy against the three cell lines whose
proliferation was inhibited in vitro. In a separate experiment,
NK-cell depletion of the SCID mice failed to abrogate the IL-21
effect in the IM-9 model, suggesting that NK-cells are not required
for the efficacy of IL-21 in this model.
[0084] Assays measuring differentiation include, for example,
measuring cell markers associated with stage-specific expression of
a tissue, enzymatic activity, functional activity or morphological
changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation
57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Biolprocesses,
161-171, 1989; all incorporated herein by reference).
Alternatively, IL-21 polypeptide itself can serve as an additional
cell-surface or secreted marker associated with stage-specific
expression of a tissue. As such, direct measurement of IL-21
polypeptide or its receptors expressed on cancer cells, or its loss
of expression in a tissue as it differentiates, can serve as a
marker for differentiation of tissues.
[0085] The classification of lymphomas most commonly used is the
REAL classification system (Ottensmeier, Chemico-Biological
Interactions 135-136:653-664, 2001.) Specific immunological markers
have been identified for classifications of lymphomas. For example,
follicular lymphoma markers include CD20+, CD3-, CD10+, CD5-; Small
lymphocytic lymphoma markers include CD20+, CD3-, CD10-, CD5+,
CD23+; marginal zone B cell lymphoma markers include CD20+, CD3-,
CD10-, CD23-; diffuse large B cell lymphoma markers include CD20+,
CD3-; mantle cell lymphoma markers include CD20+, CD3-, CD10-,
CD5+, CD23+; peripheral T cell lymphoma markers include CD20-,
CD3+; primary mediastinal large B cell lymphoma markers include
CD20+, CD3-, lymphoblastic lymphoma markers include CD20-, CD3+,
Tdt+, and Burkitt's lymphoma markers include CD20+, CD3-, CD10+,
CD5- (Decision Resourses, Non-Hodikins Lymphoma, Waltham, Mass.,
February 2002).
[0086] Primary lymphoma specimens are routinely acquired by biopsy
of nodal or extra nodal tumors in the diagnosis of lymphoma. For
some lymphoid neoplasms, in particular Chronic Lymphocytic leukemia
(CLL), the malignant cells can be acquired from the patient's
blood. One method for testing whether a specific lymphoma or
patient is amenable to treatment with IL-21 is culturing lymphoma
cells. Biopsy or blood specimens can be prepared for tissue culture
by a combination of methods well known to those skilled in the art.
For example, the samples can be prepared by mincing, teasing,
enzymatic digestion or density gradient centrifugation (ficoll)
(Jacob et al., Blood 75(5):1154-1162, 1990). The tumor cells are
then labeled with a fluorescent DNA stain such as
carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular
Probes, Eugene, Oreg.) and cultured in IL-21. The distribution of
tumor cells that have undergone 1 or more rounds of cell division
can be quatitated by flow cytometry, with the cells losing 1/2 of
their CFSE intensity with each round of replication. The proportion
of inviable cells and the number of apoptotic cells are also
analysed analyzed for the effect of IL-21 using 7-AAD and annexin-V
staining. The number of surviving cells, the distribution of CFSE
staining and the per cent of cells that are apoptotic can all be
used to determine whether IL-21 promotes or inhibits the growth and
survival of a given malignant specimen. In such an analysis the
lymphoma cells are distinguished from normal cells contaminating
the specimen by a combination of a B-cell lineage specific marker,
immunoglobulin light chain lambda and kappa specific antibody and
light scatter properties. For CLL specimens CD5 staining can also
be utilized as an aid in defining the malignant cells. As the
proportion of cells proliferating in vitro is likely to be very
low, it is essential that the tumor cells be distinguished from
normal cells. A data analysis method such as flow cytometry that
can measure multiple parameters on individual cells is
preferred.
[0087] One exemplary method for determination specific lymphoma
sensitivity to IL-21 uses biopsy or blood cells cultured in serum
free medium or in medium containing serum or plasma, preferably
fetal bovine serum or human serum, at varying doses of IL-21,
generally in a range from 0. 1 to 10 nM, and including a negative
control. At various time points, for example 1, 2, 4 and 7 days
cells are harvested and subjected to flow cytometric methods to
determine the distribution of cells that have divided 1 or more
times (CFSE intensity), the proportion of inviable cells( 7-AAD
staining; Hausner et al., J. Immunol. Methods 247 (1-2):175-186,
2001) and the number of apoptotic cells (annexin-V staining;
Lagneaux et al., Br. J. Hematol. 112 (2):344-352, 2001). The number
of surviving cells, the distribution of CFSE staining and the per
cent of cells that are apoptotic can all be used to determine
whether IL-21 promotes or inhibits the growth and survival of a
given malignant specimen. In such an analysis the lymphoma cells
are distinguished from normal cells contaminating the specimen by a
combination of a B-cell lineage specific marker, immunoglobulin
light chain lambda and kappa specific antibody and light scatter
properties. For CLL specimens CD5 staining can also be utilized as
an aid in defining the malignant cells. As the proportion of cells
proliferating in vitro may be very low, it is critical that the
tumor cells be distinguished from normal cells in the data analysis
and is why a method such as flow cytometry that can measure
multiple parameters on individual cells is useful.
[0088] Such testing of individual tumor specimens will provide
basis to choose which patients are likely to respond to IL-21
favorably and for which patients IL-21 may be contraindicated.
Patients whose malignant lymphocytes proliferate more slowly in
response to IL-21 than in control cultures or die more rapidly than
control cultures would be considered as candidates for IL-21
therapy. Similarly, a patient whose malignant cells proliferation
rate or survival is enhanced by IL-21 in vitro would, in general,
not be candidates for IL-21 therapy (except as noted below). Once
data are accumulated that demonstrates a strong correlation between
a particular type of lymphoma (for example, follicular lymphoma or
CLL) and sensitivity to IL-21 in vitro, the need to test all
patients within such a subgroup for their response to IL-21 may be
obviated, provided that no patients are found within the group that
exhibit increased proliferation in response to IL-21 in vitro.
[0089] Similarly, direct measurement of IL-21 polypeptide, or its
loss of expression in a tissue can be determined in a tissue or in
cells as they undergo tumor progression. Increases in invasiveness
and motility of cells, or the gain or loss of expression of IL-21
in a pre-cancerous or cancerous condition, in comparison to normal
tissue, can serve as a diagnostic for transformation, invasion and
metastasis in tumor progression. As such, knowledge of a tumor's
stage of progression or metastasis will aid the physician in
choosing the most proper therapy, or aggressiveness of treatment,
for a given individual cancer patient. Methods of measuring gain
and loss of expression (of either mRNA or protein) are well known
in the art and described herein and can be applied to IL-21
expression. For example, appearance or disappearance of
polypeptides that regulate cell motility can be used to aid
diagnosis and prognosis of prostate cancer (Banyard, J. and Zeffer,
B. R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of
cell motility, IL-21 gain or loss of expression may serve as a
diagnostic for lymphoid cancers.
[0090] As discussed above, the IM-9 mouse model for cancer
demonstrated that antitumor activity is not NK cell dependent.
There are several syngeneic mouse models that have been developed
to study the influence of polypeptides, compounds or other
treatments on tumor progression. In these models, tumor cells
passaged in culture are implanted into mice of the same strain as
the tumor donor. The cells will develop into tumors having similar
characteristics in the recipient mice, and metastasis will also
occur in some of the models. Appropriate tumor models for our
studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and
B16 melanoma (ATCC No. CRL-6323), amongst others. These are both
commonly used tumor lines, syngeneic to the C57BL6/J mouse, that
are readily cultured and manipulated in vitro. Tumors resulting
from implantation of either of these cell lines are capable of
metastasis to the lung in C57BL6/J mice. The Lewis lung carcinoma
model has recently been used in mice to identify an inhibitor of
angiogenesis (O'Reilly M S, et al. Cell 79: 315-328,1994). C57BL6/J
mice are treated with an experimental agent either through daily
injection of recombinant protein, agonist or antagonist or a one
time injection of recombinant adenovirus. Three days following this
treatment, 10.sup.5 to 10.sup.6 cells are implanted under the
dorsal skin. Alternatively, the cells themselves can be infected
with recombinant adenovirus, such as one expressing IL-21, before
implantation so that the protein is synthesized at the tumor site
or intracellularly, rather than systemically. The mice normally
develop visible tumors within 5 days. The tumors are allowed to
grow for a period of up to 3 weeks, during which time they may
reach a size of 1500-1800 mm.sup.3 in the control treated group.
Tumor size and body weight are carefully monitored throughout the
experiment. At the time of sacrifice, the tumor is removed and
weighed along with the lungs and the liver. The lung weight has
been shown to correlate well with metastatic tumor burden. As an
additional measure, lung surface metastases are counted. The
resected tumor, lungs and liver are prepared for histopathological
examination, immunohistochemistry, and in situ hybridization, using
methods known in the art and described herein. The influence of the
expressed polypeptide in question, e.g., IL-21, on the ability of
the tumor to recruit vasculature and undergo metastasis can thus be
assessed. In addition, aside from using adenovirus, the implanted
cells can be transiently transfected with IL-21. Use of stable
IL-21 transfectants as well as use of induceable promoters to
activate IL-21 expression in vivo are known in the art and can be
used in this system to assess IL-21 induction of metastasis.
Moreover, purified IL-21 or IL-21 conditioned media can be directly
injected in to this mouse model, and hence be used in this system.
For general reference see, O'Reilly M S, et al. Cell 79:315-328,
1994; and Rusciano D, et al. Murine Models of Liver Metastasis.
Invasion Metastasis 14:349-361, 1995.
[0091] The activity of IL-21 and its derivatives (conjugates) on
growth and dissemination of tumor cells derived from human
hematologic malignancies can be measured in vivo. Several mouse
models have been developed in which human tumor cells are implanted
into immunodeficient mice (collectively referred to as xenograft
models); see, for example, Cattan A R, Douglas E, Leuk. Res.
18:513-22, 1994 and Flavell, D J, Hematological Oncology 14:67-82,
1996. The characteristics of the disease model vary with the type
and quantity of cells delivered to the mouse, and several disease
models are known in the art. In an example of this model, tumor
cells (e.g. Raji cells (ATCC No. CCL-86)) would be passaged in
culture and about 1.times.10.sup.6 cells injected intravenously
into severe combined immune deficient (SCID) mice. Such tumor cells
proliferate rapidly within the animal and can be found circulating
in the blood and populating numerous organ systems. Therapies
designed to kill or reduce the growth of tumor cells using IL-21 or
its derivatives, agonists, conjugates or variants can be tested by
administration of IL-21 compounds to mice bearing the tumor cells.
Efficacy of treatment is measured and statistically evaluated as
increased survival within the treated population over time. Tumor
burden may also be monitored over time using well-known methods
such as flow cytometry (or PCR) to quantitate the number of tumor
cells present in a sample of peripheral blood. For example,
therapeutic strategies appropriate for testing in such a model
include direct treatment with IL-21 or related conjugates or
antibody-induced toxicity based on the interaction of IL-21 with
its receptor(s), or for cell-based therapies utilizing IL-21 or its
derivatives, agonists, conjugates or variants. The latter method,
commonly referred to as adoptive immunotherapy, would involve
treatment of the animal with components of the human immune system
(i.e. lymphocytes, NK cells, bone marrow) and may include ex vivo
incubation of cells with IL-21 with or without other
immunomodulatory agents described herein or known in the art.
[0092] The activity of IL-21 on immune (effector) cell-mediated
tumor cell destruction can be measured in vivo, using the murine
form of the IL-21 protein (SEQ ID NO:2) in syngeneic mouse tumor
models. Several such models have been developed in order to study
the influence of polypeptides, compounds or other treatments on the
growth of tumor cells and interaction with their natural host, and
can serve as models for therapeutics in human disease. In these
models, tumor cells passaged in culture or in mice are implanted
into mice of the same strain as the tumor donor. The cells will
develop into tumors having similar characteristics in the recipient
mice. For reference, see, for example, van Elsas et al., J. Exp.
Med. 190:355-66, 1999; Shrikant et al., Immunity 11:483-93, 1999;
and Shrikant et al., J. Immunol. 162:2858-66, 1999. Appropriate
tumor models for studying the activity of IL-21 on immune
(effector) cell-mediated tumor cell destruction include the B16-F10
melanoma (ATCC No. CRL-6457), and the EG.7 thymoma (ATCC No.
CRL-2113), described herein, amongst others. These are both
commonly used tumor cell lines, syngeneic to the C57BL6 mouse,
which are readily cultured and manipulated in vitro.
[0093] In an example of an in vivo model, the tumor cells (e.g.
B16-F10 melanoma (ATCC No. CRL-6475) are passaged in culture and
about 100,000 cells injected intravenously into C57BL6 mice. In
this mode of administration, B16-F10 cells will selectively
colonize the lungs. Small tumor foci are established and will grow
within the lungs of the host mouse. Therapies designed to kill or
reduce the growth of tumor cells using IL-21 or its derivatives,
agonists, conjugates or variants can be tested by administration of
compounds to mice bearing the tumor cells. Efficacy of treatment is
measured and statistically evaluated by quantitation of tumor
burden in the treated population at a discrete time point, two to
three weeks following injection of tumor cells. Therapeutic
strategies appropriate for testing in such a model include direct
treatment with IL-21 or its derivatives, agonists, conjugates or
variants, or cell-based therapies utilizing IL-21 or its
derivatives, agonists, conjugates or variants. The latter method,
commonly referred to as adoptive immunotherapy, would involve
treatment of the animal with immune system components (i.e.
lymphocytes, NK cells, dendritic cells or bone marrow, and the
like) and may include ex vivo incubation of cells with IL-2 1 with
or without other immunomodulatory agents described herein or known
in the art.
[0094] Another syngeneic mouse tumor cell line can used to test the
anti-cancer efficacy of IL-21 and to identify the immune (effector)
cell population responsible for mediating this effect. EG.7 ova is
a thymoma cell line that has been modified (transfected) to express
ovalbumin, an antigen foreign to the host. Mice bearing a
transgenic T cell receptor specific for EG.7ova are available (OT-I
transgenics, Jackson Laboratory). CD8 T cells isolated from these
animals (OT-I T cells) have been demonstrated to kill EG.7 cells in
vitro and to promote rejection of the tumor in vivo. EG.7ova cells
can be passaged in culture and about 1,000,000 cells injected
intraperitoneal into C57BL6 mice. Multiple tumor sites are
established and grow within the peritoneal cavity. Therapies
designed to kill or reduce the growth of tumor cells using IL-21 or
its derivatives, agonists, conjugates or variants can be tested by
administration of compounds to mice bearing the tumor cells. OT-I T
cells can be administered to the mice to determine if their
activity is enhanced in the presence of IL-21. Efficacy of
treatment is measured and statistically evaluated by time of
survival in the treated populations. Therapeutic strategies
appropriate for testing in such models include direct treatment
with IL-21 or its derivatives, agonists, conjugates or variants, or
cell-based therapies utilizing IL-21 or its derivatives, agonists,
conjugates or variants. Ex vivo treatment of cytotoxic
T-lymphocytes (CTL) could also be used to test the IL-21 in the
cell-based strategy.
[0095] Analysis of IL-21 efficacy for treating certain specific
types of cancers are preferably made using animals that have been
shown to correlate to other mammalian disease, particularly human
disease. After IL-21 is administered in these models evaluation of
the effects on the cancerous cells or tumors is made. Xenografts
are used for most preclinical work, using immunodeficient mice. For
example, a syngeneic mouse model for ovarian carcinoma utilizes a
C57BL6 murine ovarian carcinoma cell line stably overexpressing
VEGF16 isoform and enhanced green fluorescent protein (Zhang et
al., Am. J. Pathol. 161:2295-2309, 2002). Renal cell carcinoma
mouse models using Renca cell injections have been shown to
establish renal cell metastatic tumors that are responsive to
treatment with immunotherapeutics such as IL-12 and IL-2 (Wigginton
et al., J. of Nat. Cancer Inst. 88:38-43, 1996). A colorectal
carcinoma mouse model has been established by implanting mouse
colon tumor MC-26 cells into the splenic subcapsule of BALB/c mice
(Yao et al., Cancer Res. 63 (3):586-586-592, 2003). An
immunotherapeutic-responsive mouse model for breast cancer has been
developed using a mouse that spontaneously develops tumors in the
mammary gland and demonstrates peripheral and central tolerance to
MUC1 (Mukherjee et al., J. Immunotherapy 26:47-42, 2003). To test
the efficacy of IL-21 in prostate cancer, animal models that
closely mimic human disease have been developed. A transgenic
adenocarcinoma of the mouse prostate model (TRAMP) is the most
commonly used syngeneic model (Kaplan-Lefko et al., Prostate 55
(3):219-237, 2003; Kwon et al., PNAS 96:15074-15079, 1999; Arap et
al., PNAS 99:1527-1531, 2002).
[0096] IL-21 will be useful in treating tumorgenesis, enhancing
CTLs and NK activity, and therefore are useful in the treatment of
cancer. In addition to direct and indirect effects on CTLs and NK
cells, as shown in several tumor models described herein, IL-21
inhibits IL-4 stimulated proliferation of anti-IgM stimulated
normal B-cells and a similar effect is observed in B-cell tumor
lines suggesting that there can be therapeutic benefit in treating
patients with the IL-21 in order to induce the B cell tumor cells
into a less proliferative state.
[0097] The ligand could be administered in combination with other
agents already in use including both conventional chemotherapeutic
agents as well as immune modulators such as interferon alpha.
Alpha/beta interferons have been shown to be effective in treating
some leukemias and animal disease models, and the growth inhibitory
effects of INF-.alpha.and IL-21 are additive for at least one
B-cell tumor-derived cell line. Establishing the optimal dose level
and scheduling for IL-21 is done by a combination of means,
including the pharmacokinetics and pharmacodynamics of IL-21, the
sensitivity of human B-cell lines and primary lymphoma specimens to
IL-21 in vitro, effective doses in animal models and the toxicity
of IL-21. Optimally, to have a direct anti-tumor effect the
concentration of IL-21 in plasma should reach a level that in vitro
is maximally active against B-cell lymphoma cell lines and primary
lymphomas. In addition the optimal and minimum times of exposure to
IL-21 to elicit a growth inhibitory or apoptotic responses can be
modeled in vitro with cell lines and primary tumor cells. Direct
pharmacokinetic measurements done in primates and clinical trials
can then be used to predict theoretical doses in patients that
achieve plasma IL-21 levels that are of sufficient magnitude and
duration to achieve a biological response in patients. In addition
IL-21 stimulates a variety of responses in normal lymphocytes, such
that surrogate markers can be employed to measure the biological
activity of IL-21 on effector cells in patients.
[0098] Since lymphoma patients are treated with a variety of
chemotherapeutic drugs and drug combinations, developing a protocol
to integrate IL-21 into an existing standard treatment regimen may
result in improved therapeutic outcome. The effect of combining
chemotherapy drugs and IL-21 is primarily modeled with IL-21
sensitive human B-cell lines in vitro, measuring cell
proliferation, cell viability, and apoptosis. Time and dose
dependent response curves to chemotherapeutic drugs (e.g.
chlorambucil, etoposide, or fludaribine) are established for
individual cell lines. IL-21 is then tested over a wide range of
concentrations under suboptimal conditions of each chemotherapy
drug. The order of exposure of the cells to IL-21 versus a
chemotherapy agent may significantly affect the outcome of the
interaction with the cell line tested. As such, the IL-21 should be
introduced to the cultures in several manners to find the optimal
mode of treatment. This should include, for example, prior
treatment with IL-21 for several hours to several days (0, 4, 24,
48 and 72 hours ), followed by a wash out of IL-21 and the addition
of a sub-optimal dose/exposure time of a chemotherapy drug. After
1-3 days, analysis of the culture for cell viability, proliferation
and apoptosis is made. In a variation to the above experiment, the
IL-21 is not washed out prior to the addition of the chemotherapy
drug. The complete set of conditions to test would also include the
simultaneous treatment of cells with IL-21 and a chemotherapy drug
with varying time of IL-21 wash out, as well as delayed (from a few
hours to several days) addition of IL-21 until after exposure to a
chemotherapy drug. The timing and concentration of IL-21 exposure
that gives a maximal reduction in target cell outgrowth/viability
or maximum increase in apoptotic response will then be considered
optimal for further testing in animal models or the design of a
clinical protocol. The combination of IL-21 with chemotherapeutic
drugs in vitro using cell lines whose growth is stimulated by
IL-21, such as RPMI-1788, could be done to identify drugs that
eliminate the potential adverse affects of IL-21 that might be
encountered in a subset of patients. Such drugs would be identified
from those known to have activity against lymphomas and selected on
the basis of their ability in vitro to prevent enhanced growth and
or survival of RPMI 1788 or similarly IL-21 responsive cell lines.
In this way IL-21 therapy, when combined with selected
chemotherapeutic regimens would be of benefit to those patients
whose malignancy is sensitive to IL-21 mediated growth suppression,
while protecting any patients that might otherwise respond
unfavorably to IL-21 monotherapy.
[0099] Lymphoma patients are also treated with biologics, such as
RITUXAN.TM., IL-2 and interferon. Those biologic agents that have a
direct inhibitory effect on the tumor and are not largely dependent
on effector cells for their activity can be modeled in a manner
similar to the in vitro experiments above for their interaction
with IL-21. For example, RITUXAN.TM. binds to lymphoma cells and
can induce apoptosis directly in vitro, but is also capable of
inducing a variety of effector mechanisms such as complement
dependent cytotoxity and antibody dependent cell-mediated
cytotoxicity (ADCC). Therefore, it is possible to define conditions
in vitro in which IL-21 and RITUXAN.TM. interact in synergy to
inhibit lymphoma growth or stimulate apoptosis. The use of a
xenogeneic human lymphoma model in SCID mice has the potential to
measure a broader range of potential interactions that involve host
effector mechanisms between RITUXAN.TM. (or some other biologic
agent) and IL-21. To determine if there is significant synergy
between IL-21 and another anti-tumor biologic in a xenogeneic
lymphoma SCID mouse model, IL-21 and the other biologic are tested
under conditions that yield marginal therapeutic results with
either agent alone.
[0100] IL-21 and IL-2 exhibit synergy in their effects on NK-cells
in vitro with respect to IFN-.gamma. production and proliferation.
In addition, high dose IL-2 therapy is highly toxic and requires
extensive hospitalization. Many low dose regimens of IL-2 have been
tested, and found to be well tolerated, but with little evidence of
anti-tumor efficacy (Atkins, Semin. Oncol. 29 (3 Suppl. 7):12,
2002). The combination of low dose IL-2 with IL-21 therefore may be
clinically useful by augmenting the immune system stimulation of
low dose IL-2 while providing a direct anti-lymphoma effect. The
effects of combining IL-2 and IL-21 are studied in mouse syngeneic
lymphoma models or in SCID mouse xenogeneic human lymphoma models
as described herein. The relative effects on effector cell
activation and toxicity of combining IL-2 and IL-21 at different
doses can be determined in normal primates to optimize a dosing
level and schedule to avoid the need to hospitalize patients.
[0101] For those patients whose malignant lymphocytes are
stimulated to proliferate in vitro in response to IL-21, a course
of IL-21 could be contraindicated (in the absence of combination
with other drugs as discussed above) unless the malignant cells
have a very low turn over rate in vivo, such as CLL. The relatively
quiescent state of CLL cells may be related to the resistance of
this disease to chemotherapy. In such cases, patients could be
treated by pulsing them with IL-21 just prior to the administration
of chemotherapeutic drug(s). The optimal timing of dosing of IL-21
and chemotherapy could be modeled in vitro to predict how long
after exposure to IL-21 the malignant cells become maximally
sensitive to specific chemotherapeutic drugs.
[0102] The present invention provides a method of reducing
proliferation of a neoplastic B or T cells comprising administering
to a mammal with a B or T cell lymphoma an amount of a composition
of IL-21 sufficient to reduce proliferation of the B or T lymphoma
cells. In other embodiments, the composition can comprise at least
one other cytokine selected from the group consisting of IL-2,
IL-15, IL-4, IL-18, GM-CSF, Flt3 ligand, interferon, or stem cell
factor.
[0103] In another aspect, the present invention provides a method
of reducing proliferation of a neoplastic B or T cells comprising
administering to a mammal with a B or T cell neoplasm an amount of
a composition of IL-21 antagonist sufficient to reducing
proliferation of the neoplastic B or T cells. In other embodiments,
the composition can comprise at least one other cytokine selected
from the group consisting of IL-2, IL-15, IL-4, IL-18, GM-CSF, Flt3
ligand, interferon, or stem cell factor. Furthermore, the IL-21
antagonist can be a ligand/toxin fusion protein.
[0104] A IL-21-saporin fusion toxin, or other IL-21-toxin fusion,
can be employed against a similar set of leukemias and lymphomas,
extending the range of leukemias that can be treated with IL-21 .
Moreover, such IL-21-toxin fusions can be employed against other
cancers wherein IL-21 binds its receptors. Fusion toxin mediated
activation of the IL-21 receptor provides two independent means to
inhibit the growth of the target cells, the first being identical
to the effects seen by the ligand alone, and the second due to
delivery of the toxin through receptor internalization. The
lymphoid restricted expression pattern of the IL-21 receptor
suggests that the ligand-saporin conjugate can be tolerated by
patients.
[0105] When treatment for malignancies includes allogeneic bone
marrow or stem cell transplantation, IL-21 can be valuable in
enhancement of the graft-vs-tumor effect. IL-21 stimulates the
generation of lytic NK cells from marrow progenitors and stimulates
the proliferation of T-cells following activation of the antigen
receptors. Therefore, when patients receive allogenic marrow
transplants, IL-21 will enhance the generation of anti-cancer
responses, with or without the infusion of donor lymphocytes.
[0106] Modern methods for cancer immunotherapy are based on the
principle that the immune system can detect and defend against
spontaneous tumors. Evidence supporting the concept of
"Immunological surveillance" (see, Burnet FM Lancet 1: 1171-4,
1967), comes in part from epidemiological studies indicating that
the incidence of cancer increases in patients that are
immunocomprised by disease, such as infection (see, Klein G. Harvey
Lect. 69:71-102, 1975; and Kuper et al., J. Intern. Med.
248:171-83, 2000), or following medical interventions such as bone
marrow ablation (see, Birkeland et al., Lancet 355:1886-7, 2000;
and Penn I, Cancer Detect Prev. 18:241-52, 1994). Experiments
performed in gene-targeted mice also show that the immune system
modulates susceptibility to spontaneous tumors in aged mice (see,
Smyth, M. et al., J. Exp. Med. 192:755-760, 2000; and Davidson, W.
et al., J. Exp. Med. 187: 1825-1838, 1998) or following exposure to
chemical carcinogens (see, Peng, S et al., J. Exp. Med. 184:
1149-1154, 1996; Kaplan, D. et al., Proc. Nat. Acad Sci. USA
95:7556-7561, 1998; and Shankaran V. et al., Nature 410:1107-1111,
2001). Proof that immune recognition of tumors occurs frequently in
tumor bearing hosts comes from the identification of T-cells that
are reactive to a broad range of tumor associated antigens
including differentiation antigens, mutational antigens,
tissue-specific antigens, cancer-testis antigens, self antigens
that are over expressed in tumors, and viral antigens (Boon T. et
al., Immunol. Today 18:267-8, 1997.). In addition, B-cells are
known to produce high titers of circulating IgG antibodies that
recognize these same classes of tumor antigens (Stockert E. et al.,
J. Exp. Med. 187:1349-54, 1998; . Sahin U et al., Curr. Opin.
Immunol. 9:709-16, 1997; and Jager, E. et al., Proc. Nat. Acad.
Sci. USA 97:12198-12203, 2000), and NK cells have been isolated
that can recognize and kill tumor cells that express various
stress-related genes (Bauer, S et al., Science 285:727-729,
1999).
[0107] The concept that immunotherapy can be an effective method
for treating cancer is firmly established in experimental animal
models, and while the methodologies are much less advanced for
human subjects, there is a strong suggestion that the immune system
can be stimulated to reject established disease. The very first
attempt at cancer immunotherapy was reported by William Coley in
1893 who, using extracts of pyrogenic bacteria, achieved
anti-cancer responses most likely through the induction of systemic
inflammation and cell-mediated immunity (Coley WB. The treatment of
malignant tumors by repeated inoculations of erysipelas. With a
report of ten original cases. 1893, Clin Orthop. 262:3-11, 1991).
In more modern times five generalized strategies have been employed
to increase the numbers of effector cells and/or modulate their
anti-cancer activity (reviewed in Rosenberg, SA. (Ed.), Principles
and practice of the biologic therapy of cancer., 3.sup.rd edition,
Lippincott Williams & Wilkins, Philadelphia, Pa., 2000):
cytokine therapy, cell transfer therapy, monoclonal antibody
therapy, cancer vaccines, and gene therapy. To date, each method
has shown effectiveness in mediating an anti-cancer response
although the durability of these responses, with a few exceptions,
is mostly temporary. This fact reflects our limited understanding
of tumor immunology and argues that improvements in the technology
await the utilization of previously unrecognized elements of the
anti-cancer response. The present invention provides such an
element to improve our understanding of tumor immunology as well as
provide polypeptides that are therapeutically useful in treating
and preventing human cancers.
[0108] One requirement for achieving sustained immunity and durable
clinical responses is the amplification in the level, i.e., in the
numbers and activity of the cells that mediate tumor killing. Thus,
new factors that mediate their effects on lymphocytes including
cytotoxic T-cells (CTLs), NK cells, and B-cells, as well myeloid
cells such as neutrophils and monocytic cells will improve
anti-cancer activity. IL-21 is a product of activated CD4.sup.+
"helper" T-cells which are required for both humoral and
cell-mediated immunity and for sustaining long-term memory to
antigenic re-challenge (U.S. Pat. No. 6,307,024; Parrish-Novak J et
al., Nature 408:57-63, 2000). The receptor for IL-21 is expressed
on cells that mediate anti-cancer responses and previous
experiments have shown that IL-21 can stimulate the proliferation
of these cell types in vitro (commonly-owned WIPO Publication No.s
WO 0/17235 and WO 01/77171). Additional experiments affirm these
IL-21 activities in vivo.
[0109] IL-21 polypeptides for the methods of the present invention
are shown to stimulate CTL and NK cells against tumors in vivo in
animal models resulting in decreased tumor burden and tumor cells,
and increased survival. IL-21 can hence be used in therapeutic
anti-cancer applications in humans. As such, IL-21 anti-cancer
activity is useful in the treatment and prevention of human
cancers. Such indications include but are not limited to the
following: Carcinomas (epithelial tissues), Sarcomas of the soft
tissues and bone (mesodermal tissues), Adenomas (glandular
tissues), cancers of all organ systems, such as liver (hepatoma)
and kidney (renal cell carcinomas), CNS (gliomas, neuroblastoma),
and hematological cancers, viral associated cancers (e.g.,
associated with retroviral infections, HPV, hepatitis B and C, and
the like), lung cancers, endocrine cancers, gastrointestinal
cancers (e.g., biliary tract cancer, liver cancer, pancreatic
cancer, stomach cancer and colorectal cancer), genitourinary
cancers (e.g., prostate cancer bladder cancer, renal cell
carcinoma), gynecologic cancers (e.g., uterine cancer, cervical
cancer, ovarian cancer) breast, and other cancers of the
reproductive system, head and neck cancers, and others. Of
particular interest are hematopoietic cancers, including but not
limited to, lymphocytic leukemia, myeloid leukemia, Hodgkin's
lymphoma, Non-Hodgkins lymphomas, chronic lymphocytic leukemia, and
other leukemias and lymphomas. Moreover IL-21 can be used
therapeutically in cancers of various non-metastatic as wells as
metastatic stages such as "Stage 1" Localized (confined to the
organ of origin); "Stage 2" Regional; "Stage 3" Extensive; and
"Stage 4" Widely disseminated cancers. In addition, IL-21 can be
used in various applications for cancer, immunotherapy, and in
conjunction with chemotherapy and the like.
[0110] Administration of IL-21 using the methods of the present
invention will result in a tumor response. While each protocol may
define tumor response accessments differently, exemplary guidelines
can be found in Clinical Research Associates Manual, Southwest
Oncology Group, CRAB, Seattle, Wash., Oct. 6, 1998, updated August
1999. According to the CRA Manual (see, chapter 7 "Response
Accessment"), tumor response means a reduction or elimination of
all measurable lesions or metastases. Disease is generally
considered measurable if it comprises bidimensionally measurable
lesions with clearly defined margins by medical photograph or
X-ray, computerized axial tomography (CT), magnetic resonance
imaging (MRI), or palpation. Evaluable disease means the disease
comprises unidimensionally measurable lesions, masses with margins
not clearly defined, lesion with both diameters less than 0.5 cm,
lesions on scan with either diameter smaller than the distance
between cuts, palpable lesions with diameter less than 2 cm, or
bone disease. Non-evaluable disease includes pleural effusions,
ascites, and disease documented by indirect evidence. Previously
radiated lesions which have not progressed are also generally
considered non-evaluable.
[0111] The criteria for objective status are required for protocols
to access solid tumor response. A representative criteria includes
the following: (1) Complete Response (CR) defined as complete
disappearance of all measurable and evaluable disease. No new
lesions. No disease related symptoms. No evidence of non-evaluable
disease; (2) Partial Response (PR) defined as greater than or equal
to 50% decrease from baseline in the sum of products of
perpendicular diameters of all measureable lesions. No progression
of evaluable disease. No new lesions. Applies to patients with at
least one measurable lesion; (3) Progression defined as 50% or an
increase of 10 cm.sup.2 in the sum of products of measurable
lesions over the smallest sum observed using same techniques as
baseline, or clear worsening of any evaluable disease, or
reappearance of any lesion which had disappeared, or appearance of
any new lesion, or failure to return for evaluation due to death or
deteriorating condition (unless unrelated to this cancer); (4)
Stable or No Response defined as not qualifying for CR, PR, or
Progression. (See, Clinical Research Associates Manual, supra.)
[0112] Examples of methods for using IL-21 in the treatment of
cancer include, but are not limited to, the following:
[0113] 1) IL-21 can be used as a single agent for direct inhibitory
activity against tumors that express the IL-21 receptor (U.S. Pat.
No. 6,307,024; WIPO Publication No.s WO 0/17235 and WO 01/77171).
Such activity is shown herein. Administration in a pharmaceutical
vehicle for therapeutic use can be achieved using methods in the
art and described herein.
[0114] 2) IL-21 can be conjugated to a toxic compound that binds
and kills tumor cells that express IL-21 receptor such as B-cell
lymphomas, T-cell lymphomas and NK cell lymphomas. The toxic
compound can be a small molecule drug like calichaemicin used in a
manner similar to the anti-CD33 antibody+drug conjugate,
MYLOTARG.TM., that is used to treat acute myelogeous leukemia (for
example, See, Sievers E L et al., J Clin Oncol. 19:3244-54, 2001;
and Bernstein I D Clin. Lymphoma Suppl 1:S9-S11, 2002); or a
radioisotope like .sup.125I (Kaminski M S, et al. J. Clin. Oncol.
19:3918-28, 2001) or .sup.90Y (Reviewed in Gordon L I et al.,
Semin. Oncol. (1 Suppl 2):87-92, 2002) that has been attached to an
anti-CD20 antibody used for the treatment of Non-Hogkin's
lymophoma; or a naturally occurring protein toxin such as Ricin A
(Lynch T J Jr, et al., J. Clin. Oncol. 15:723-34, 1997) or
diphtheria B toxin that was made as a fusion protein with IL-2 for
the treatment of cutaneous T-cell lymphoma (Talpur R et al., Leuk.
Lymphoma 43:121-6, 2002). The attachment of these toxic compounds
to IL-21 might occur through chemical conjugation (Rapley R. Mol.
Biotechnol. 3:139-54, 1995) or genetic recombination (Foss F M.
Clin. Lymphoma Suppl 1:S27-31, 2000). Such toxin conjugates with
IL-21, for example IL-21-saporin conjugates, are shown to kill
various tumors in vivo and in vitro (U.S. Pat. No. 6,307,024; and
described herein).
[0115] IL-21 can be used as an immunostimulatory agent for cancer
monotherapy. A variety of cytokines such as IL-2, IL-4, IL-6,
IL-12, IL-15, and interferon, are known to stimulate anti-cancer
responses in animal models via stimulation of the immune system
(reviewed in Rosenberg, SA ibid.). Moreover IL-21 is shown to also
stimulate the immune system (U.S. Pat. No. 6,307,024; and described
herein). Cytokine monotherapy is an accepted practice for human
cancer patients. For example, the use of IL-2 and IFN-.alpha. are
used for the treatments of metastatic melanoma and renal cell
carcinoma (e.g., see, Atkins M B et al., J. Clin. Oncol.
17:2105-16, 1999; Fyfe G et al., J. Clin. Oncol. 13:688-96, 1995;
and Jonasch E, and Haluska F G, Oncologist 6:34-55, 2001). The
mechanism of action of these cytokines includes, but is not limited
to, an enhancement of a Th1 cell-mediated responses including
direct tumor cell killing by CD8+ T-cells and NK cells. IL-21 is
shown to similarly enhance Th1 cell-mediated responses including
direct tumor cell killing by CTLs, e.g., CD8+ T-cells, and NK cells
in vivo and in vitro as described herein. Thus, IL-21 of the
present invention can be used therapeutically or clinically to
actively kill tumor cells in human disease, and to regulate these
activities, as well as in additional anti-cancer responses.
[0116] 4) IL-21 can be used as an immunostimulatory agent in
combination with chemotherapy, radiation, and myeloablation. In
addition to working alone to boost anti-cancer immunity in
patients, IL-21 can work in synergy with standard types of
chemotherapy or radiation. For instance, in preclinical models of
lymphoma and renal cell carcinoma, the combination of IL-2 with
doxorubicin (Ehrke M J et al., Cancer Immunol. Immunother.
42:221-30, 1996), or the combinations of IL-2 (Younes E et al.,
Cell Immunol. 165:243-51, 1995) or IFN-.alpha. (Nishisaka N et al.,
Cytokines Cell Mol Ther. 6:199-206, 2000) with radiation provided
superior results over the use of single agents. In this setting,
IL-21 can further reduce tumor burden and allow more efficient
killing by the chemotherapeutic. Additionally, lethal doses of
chemotherapy or radiation followed by bone marrow transplantation
or stem cell reconstitution could reduce tumor burden to a
sufficiently small level (ie. minimal residual disease) to better
allow an IL-21 mediated anti-cancer effect. Examples of this type
of treatment regimen include the uses of IL-2 and IFN-.alpha. to
modify anti-cancer responses following myeloablation and
transplantation (Porrata L F et al., Bone Marrow Transplant.
28:673-80, 2001; Slavin S, and Nagler A. Cancer J. Sci. Am. Suppl
1:S59-67, 1997; and Fefer A et al., Cancer J. Sci. Am. Suppl
1:S48-53, 1997). In the case of lymphoma and other cancers,
depending on when IL-21 is used relative to the chemotherapeutic
agents, IL-21 may be employed to directly synergize with the
chemotherapeutic agent's effect on the tumor cells or alternatively
employed after the chemotherapy to stimulate the immune system.
Those skilled in the art would design a protocol to take advantage
of both possibilities.
[0117] 5) IL-21 can be used as a tissue protective agent in
combination with standard forms of chemotherapy or methods that
ablate bone marrow. IL-21 regulates the proliferation and
differentiation of cells. As a result, IL-21 can protect various
tissues and organs from the toxicities associated with commonly
used chemotherapies and radiation. As an example, gut epithelium
expresses IL-15 receptor and experiments in animal models show that
IL-15 protects intestinal epithelium from chemotherapy induced
toxicity and prevents morbidity (Shinohara H et al., Clin. Cancer
Res. 5:2148-56, 1999; Cao S et al., Cancer Res. 58:3270-4, 1998;
and Cao S et al., Cancer Res. 58: 1695-9, 1998). In addition to
protecting against damage, the proliferative effects of IL-21 can
accelerate tissue regeneration following drug-induced toxicity.
Relevant examples of this type of activity include the enhanced
reconstitution of the immune system stimulated by IL-7 following
bone marrow transplantation (Alpdogan O et al., Blood 98:2256-65,
2001; and Mackall C L et al., Blood 97:1491-7, 2001) and the use of
G-CSF to treat neutropenia following chemotherapy (Lord, B I et
al., Clin. Cancer Res. 7:2085-90, 2001; and Holmes FA et al., J.
Clin. Oncol. 20:727-31, 2002). Because IL-21 is shown to enhance
proliferation and differentiation of hematopoietic and lymphoid
cells, IL-21 of the present invention can be used therapeutically
or clinically to aid in recovery as well as enhance the
chemotherapeutic dosage schemes upon administration of
chemotherapeutic agents in human disease.
[0118] 6) IL-21 can be used in combination with other
immunomodulatory compounds including various cytokines and
co-stimulatory/inhibitory molecules. The immunostimulatory activity
of IL-21 in mediating an anti-cancer response can be enhanced in
patients when IL-21 is used with other classes of immunomodulatory
molecules. These could include, but are not limited to, the use of
additional cytokines. For instance, the combined use of IL-2 and
IL-12 shows beneficial effects in T-cell lymphoma, squamous cell
carcinoma, and lung cancer (Zaki M H et al., J. Invest. Dermatol.
118:366-71, 2002; Li D et al., Arch. Otolaryngol. Head Neck Surg.
127:1319-24, 2001; and Hiraki A et al., Lung Cancer 35:329-33,
2002). In addition IL-21 could be combined with reagents that
co-stimulate various cell surface molecules found on immune-based
effector cells, such as the activation of CD137 (Wilcox R A et al.,
J. Clin. Invest. 109:651-9, 2002) or inhibition of CTLA4 (Chambers
C A et al., Ann. Rev. Immunol. 19:565-94, 2001). Alternatively,
IL-21 could be used with reagents that induce tumor cell apoptosis
by interacting with TRAIL-related receptors (Takeda K et al., J.
Exp. Med. 195:161-9, 2002; and Srivastava R K, Neoplasia 3:535-46,
2001). Such reagents include TRAIL ligand, TRAIL ligand-Ig fusions,
anti-TRAIL antibodies, and the like.
[0119] 7) IL-21 can be used in combination with Monoclonal Antibody
Therapy. Treatment of cancer with monoclonal antibodies is becoming
a standard practice for many tumors including Non-Hodgkins lymphoma
(RITUXAN.TM.), forms of leukemia (MYLOTARG.TM.), breast cell
carcinoma (HERCEPTIN.TM.), and colon carcinoma (ERBITUX.TM.). One
mechanism by which antibodies mediate an anti-cancer effect is
through a process referred to as antibody-dependent cell-mediated
cytotoxicity (ADCC) in which immune-based cells including NK cells,
macrophages and neutrophils kill those cells that are bound by the
antibody complex. Due to its immunomodulatory activity, IL-21 can
be used to enhance the effectiveness of antibody therapy. Examples
of this type of treatment paradigm include the combination use of
RITUXAN.TM. and either IL-2, IL-12, or IFN-.alpha. for the
treatment of Hodgkin's and Non-Hodgkin's lymphoma (Keilholz U et
al., Leuk. Lymphoma 35:641-2, 1999; Ansell S M et al., Blood
99:67-74, 2002; Carson W E et al., Eur. J. Immunol. 31:3016-25,
2001; and Sacchi S et al., Haematologica 86:951-8, 2001).
Similarly, Because IL-21 is shown to enhance proliferation and
differentiation of hematopoietic and lymphoid cells, as well as NK
cells, IL-21 of the present invention can be used therapeutically
or clinically to enhance the enhance the activity and effectiveness
of antibody therapy in human disease.
[0120] 8) IL-21 can be used in combination with cell adoptive
therapy. One method used to treat cancer is to isolate anti-cancer
effector cells directly from patients, expand these in culture to
very high numbers, and then to reintroduce these cells back into
patients. The growth of these effector cells, which include NK
cells, LAK cells, and tumor-specific T-cells, requires cytokines
such as IL-2 (Dudley M E et al., J. Immunother. 24:363-73, 2001).
Given its growth stimulatory properties on lymphocytes, IL-21 could
also be used to propagate these cells in culture for subsequent
re-introduction into patients in need of such cells. Following the
transfer of cells back into patients, methods are employed to
maintain their viability by treating patients with cytokines such
as IL-2 (Bear H D et al., Cancer Immunol. Immunother. 50:269-74,
2001; and Schultze J L et al., Br. J. Haematol. 113:455-60, 2001).
Again, IL-21 can be used following adoptive therapy to increase
effector cell function and survival.
[0121] 9) IL-21 can be used in combination with tumor vaccines. The
major objective of cancer vaccination is to elicit an active immune
response against antigens expressed by the tumor. Numerous methods
for immunizing patients with cancer antigens have been employed,
and a variety of techniques are being used to amplify the strength
of the immune response following antigen delivery (reviewed in
Rosenberg, S A ibid). Methods in which IL-21 can be used in
combination with a tumor vaccine include, but are not limited to,
the delivery of autologous and allogeneic tumor cells that either
express the IL-21 gene or in which IL-21 is delivered in the
context of a adjuvant protein. Similarly, IL-21 can be delivered in
combination with injection of purified tumor antigen protein, tumor
antigen expressed from injected DNA, or tumor antigen peptides that
are presented to effector cells using dendritic cell-based
therapies. Examples of these types of therapies include the use of
cytokines like IL-2 in the context of vaccination with modified
tumor cells (Antonia S J et al., J. Urol. 167:1995-2000, 2002; and
Schrayer D P et al., Clin. Exp. Metastasis 19:43-53, 2002), DNA
Niethammer A G et al., Cancer Res. 61:6178-84, 2001), and dendritic
cells (Shimizu K et al., Proc. Nat. Acad. Sci U S A 96:2268-73,
1999). Similarly, IL-21 can be used as an anti-cancer vaccine
adjuvant.
[0122] 10) IL-21 can be used in the context of gene therapy. Gene
therapy can be broadly defined as the transfer of genetic material
into a cell to transiently or permanently alter the cellular
phenotype. Numerous methods are being developed for delivery of
cytokines, tumor antigens, and additional co-stimulatory molecules
via gene therapy to specific locations within tumor patients
(reviewed in Rosenberg, S A ibid). These methodologies could be
adapted to use IL-21 DNA or RNA, or IL-21 could be used as a
protein adjuvant to enhance immunity in combination with a gene
therapy approach as described herein.
[0123] The tissue distribution of a receptor for a given cytokine
offers a strong indication of the potential sites of action of that
cytokine. Northern analysis of IL-21 receptor revealed transcripts
in human spleen, thymus, lymph node, bone marrow, and peripheral
blood leukocytes. Specific cell types were identified as expressing
IL-21 receptors, and strong signals were seen in a mixed lymphocyte
reaction (MLR) and in the Burkitt's lymphoma Raji. The two
monocytic cell lines, THP-1 (Tsuchiya et al., Int. J. Cancer
26:171-176, 1980) and U937 (Sundstrom et al., Int. J. Cancer
17:565-577, 1976), were negative.
[0124] IL-21 receptor is expressed at relatively high levels in the
MLR, in which peripheral blood mononuclear cells (PBMNC) from two
individuals are mixed, resulting in mutual activation. Detection of
high levels of transcript in the MLR but not in resting T or B cell
populations suggests that IL-21 receptor expression may be induced
in one or more cell types during activation. Activation of isolated
populations of T and B cells can be artificially achieved by
stimulating cells with PMA and ionomycin. When sorted cells were
subjected to these activation conditions, levels of IL-21 receptor
transcript increased in both cell types, supporting a role for this
receptor and IL-21 in immune responses, especially in autocrine and
paracrine T and B cell expansions during activation. IL-21 may also
play a role in the expansion of more primitive progenitors involved
in lymphopoiesis.
[0125] IL-21 receptor was found to be present at low levels in
resting T and B cells, and was upregulated during activation in
both cell types. Interestingly, the B cells also down-regulate the
message more quickly than do T cells, suggesting that amplitude of
signal and timing of quenching of signal are important for the
appropriate regulation of B cell responses.
[0126] IL-21 in concert with IL-15 expands NK cells from bone
marrow progenitors and augments NK cell effector function. IL-21
also co-stimulates mature B cells stimulated with anti-CD40
antibodies, but inhibits B cell proliferation to signals through
IgM. IL-21 enhances T cell proliferation in concert with a signal
through the T cell receptor, and overexpression in transgenic mice
leads to lymphopenia and an expansion of monocytes and
granulocytes, as described herein.
[0127] IL-21 polypeptides and proteins can also be used ex vivo,
such as in autologous marrow culture. Briefly, bone marrow is
removed from a patient prior to chemotherapy or organ transplant
and treated with IL-21, optionally in combination with one or more
other cytokines. The treated marrow is then returned to the patient
after chemotherapy to speed the recovery of the marrow or after
transplant to suppress graft vs. Host disease. In addition, the
proteins of the present invention can also be used for the ex vivo
expansion of marrow or peripheral blood progenitor (PBPC) cells.
Prior to treatment, marrow can be stimulated with stem cell factor
(SCF) to release early progenitor cells into peripheral
circulation. These progenitors can be collected and concentrated
from peripheral blood and then treated in culture with IL-21,
optionally in combination with one or more other cytokines,
including but not limited to SCF, IL-2, IL-4, IL-7, IL-15, IL-18,
or interferon, to differentiate and proliferate into high-density
lymphoid cultures, which can then be returned to the patient
following chemotherapy or transplantation.
[0128] The present invention provides a method for expansion of
hematopoietic cells and hematopoietic cell progenitors comprising
culturing bone marrow or peripheral blood cells with a composition
comprising an amount of IL-21 sufficient to produce an increase in
the number of lymphoid cells in the bone marrow or peripheral blood
cells as compared to bone marrow or peripheral blood cells cultured
in the absence of IL-21. In other embodiments, the hematopoietic
cells and hematopoietic progenitor cells are lymphoid cells. In
another embodiment, the lymphoid cells are NK cells or cytotoxic T
cells. Furthermore, the composition can also comprise at least one
other cytokine selected from the group consisting of IL-2, IL-15,
IL-4, GM-CSF, Flt3 ligand and stem cell factor.
[0129] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Mouse IL-21 is Active in Mouse Bone Marrow Assay
A. Isolation of Non-adherent Low Density Marrow Cells:
[0130] Fresh mouse femur aspirate (marrow) was obtained from 6-10
week old male Balb/C or C57BL/6 mice. The marrow was then washed
with RPMI+10% FBS (JRH, Lenexa KS; Hyclone, Logan Utah.) and
suspended in RPMI+10% FBS as a whole marrow cell suspension. The
whole marrow cell suspension was then subjected to a density
gradient (Nycoprep, 1.077, Animal; Gibco BRL) to enrich for low
density, mostly mononuclear, cells as follows: The whole marrow
cell suspension (About 8 ml) was carefully pipetted on top of about
5 ml Nycoprep gradient solution in a 15 ml conical tube, and then
centrifuged at 600.times. g for 20 minutes. The interface layer,
containing the low density mononuclear cells, was then removed,
washed with excess RPMI+10% FBS, and pelleted by centrifugation at
400.times. g for 5-10 minutes. This pellet was resuspended in
RPMI+10% FBS and plated in a T-75 flask at approximately 10.sup.6
cells/ml, and incubated at 37.degree. C. 5% CO.sub.2 for
approximately 2 hours. The resulting cells in suspension were
Non-Adherent Low Density (NA LD) Marrow Cells.
B. 96-Well Assay
[0131] NA LD Mouse Marrow Cells were plated at 25,000 to 45,000
cells/well in 96 well tissue culture plates in RPMI+10% FBS+1 ng/mL
mouse Stem Cell Factor (mSCF) (R&D Systems, Minneapolis,
Minn.), plus 5% conditioned medium from one of the following: (1)
BHK 570 cells expressing mouse IL-21 (U.S. Pat. No. 6,307,024), (2)
BHK 570 cells expressing human IL-21 (U.S. Pat. No. 6,307,024), or
(3) control BHK 570 cells containing vector and not expressing
either Ligand. These cells were then subjected to a variety of
cytokine treatments to test for expansion or differentiation of
hematopoietic cells from the marrow. To test, the plated NA LD
mouse marrow cells were subjected to human Interleukin-15 (hIL-15)
(R&D Systems), or one of a panel of other cytokines (R&D
Systems). Serial dilution of hIl-15, or the other cytokines, were
tested, with 2-fold serial dilution from about 50 ng/ml down to
about 6025 ng/ml concentration. After 8 to 12 days the 96-well
assays were scored for cell proliferation by Alamar blue assay as
described in U.S. Pat. No. 6,307,024.
C. Results from the 96-well NA LD Mouse Marrow Assay
[0132] Conditioned media from the BHK cells expressing both mouse
and human IL-21 acted in synergy with hIL-15 to promote the
expansion of a population of hematopoietic cells in the NA LD mouse
marrow. This expansion of hematopoietic cells was not shown with
control BHK conditioned medium plus IL-15. The population
hematopoietic cells expanded by the mouse IL-21 with hIL-15, and
those hematopoietic cells expanded by the human IL-21 with hIL-15,
were further propagated in cell culture. These hematopoietic cells
were stained with a Phycoerythrin labeled anti-Pan NK cell antibody
(Pharmingen) and subjected to flow cytometry analysis, which
demonstrated that the expanded cells stained positively for this
natural killer (NK) cell marker.
[0133] The same 96-well assay was run, using fresh human marrow
cells bought from Poietic Technologies, Gaithersburg, Md. Again, in
conjunction with IL-15, the mouse and human IL-21 expanded a
hematopoietic cell population that stained positively for the NK
cell marker using the antibody disclosed above.
Example 2
IL-21 Transgenic Mice
A. Generation of transgenic mice expressing human and mouse
IL-21
[0134] DNA fragments from transgenic vectors (U.S. Pat. No.
6,307,024) containing 5' and 3' flanking sequences of the
respective promoter (MT-1 liver-specific promoter (mouse IL-21
(U.S. Pat. No. 6,307,024) or lymphoid specific LCK promoter (mouse
and human IL-21 (U.S. Pat. No. 6,307,024), the rat insulin II
intron, IL-21 cDNA and the human growth hormone poly A sequence
were prepared and used for microinjection into fertilized B6C3fl
(Taconic, Germantown, N.Y.) murine oocytes, using a standard
microinjection protocol. See, Hogan, B. et al., Manipulating the
Mouse Embryo. A Laboratory Manual Cold Spring Harbor Laboratory
Press, 1994.
[0135] Eight transgenic mice expressing human IL-21 from the
lymphoid-specific E.mu.LCK promoter were identified among 44 pups.
Four of these were pups that died and 4 grew to adulthood.
Expression levels were fairly low in these animals. Twenty
transgenic mice expressing mouse IL-21 from the lymphoid-specific
E.mu.LCK promoter were identified among 77 pups. All 20 grew to
adulthood. Expression levels were fairly low in these animals.
Three transgenic mice expressing mouse IL-21 from the
liver-specific MT-1 promoter were identified among 60 pups. Two of
these pups died and 1 grew to adulthood. Expression levels were
fairly low in these animals. Tissues were prepared and
histologically examined as describe below.
B. Microscopic Evaluation of Tissues from Transgenic Mice
[0136] Spleen, thymus, and mesenteric lymph nodes were collected
and prepared for histologic examination from transgenic animals
expressing human and mouse IL-21 (Example 2A). Other tissues which
were routinely harvested included the following: Liver, heart,
lung, kidney, skin, mammary gland, pancreas, stomach, small and
large intestine, brain, salivary gland, trachea, espohogus,
adrenal, pituitary, reproductive tract, accessory male sex glands,
skeletal muscle including peripheral nerve, and femur with bone
marrow. The tissues were harvested from a neonatal pup which died
unexpectedly, and several adult transgenic mice, as described
below. Samples were fixed in 10% buffered formalin, routinely
processed, embedded in paraffin, sectioned at 5 microns, and
stained with hematoxylin and eosin. The slides were examined and
scored as to severity of tissue changes (0=none, 1=mild,
2=moderate, 3=severe) by a board certified veterinary pathologist
blinded to treatment.
[0137] The pup and 2 female adult mice expressing the human IL-21,
and 3 of the 6 male adult mice expressing the mouse IL-21 showed
inflammatory infiltrates in many of the tissues examined. The
organs affected varied somewhat from mouse to mouse. The
inflammatory infiltrate was composed primarily of neutrophils and
macrophages in varying numbers and proportions and was generally
mild to moderate degree in severity. Moreover, these animals showed
changes in lymphoid organs, including moderate to severe
lymphopenia in the spleen and thymus (human and mouse IL-21
transgenics); and severe lymphopenia (human IL-21 transgenics), or
mild to severe suppurative to pyogranulomatous lymphadenitis (mouse
IL-21 transgenics) in lymph nodes. In addition, increased
extramedullary hematopoiesis was evident in the spleens. These
changes were not observed in age-matched control mice.
C. Flow Cytometric Analysis of Tissues from Transgenic Mice over
Expressing IL-21
[0138] Transgenic animals over expressing either human or mouse
zalpha 11 ligand (Example 2A) were sacrificed for flow cytometric
analysis of peripheral blood, thymus, lymph node, bone marrow, and
spleen.
[0139] Cell suspensions were made from spleen, thymus and lymph
nodes by teasing the organ apart with forceps in ice cold culture
media (500 ml RPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5
ml 100.times. L-glutamine (Gibco BRL. Grand Island, N.Y.); 5 ml
100.times. Na Pyruvate (Gibco BRL); 5 ml 100.times. Penicillin,
Streptomycin, Neomycin (PSN) (Gibco BRL) and then gently pressing
the cells through a cell strainer (Falcon, VWR Seattle, Wash.).
Peripheral blood (200 ml) was collected in heparinized tubes and
diluted to 10 ml with HBSS containing 10U Heparin/ml. Erythrocytes
were removed from spleen and peripheral blood preparations by
hypotonic lysis. Bone marrow cell suspensions were made by flushing
marrow from femurs with ice cold culture media. Cells were counted
and tested for viability using Trypan Blue (GIBCO BRL,
Gaithersburg, Md.). Cells were resuspended in ice cold staining
media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) at a
concentration of ten million per milliliter. Blocking of Fc
receptor and non-specific binding of antibodies to the cells was
achieved by adding 10% normal goat sera and Fc Block (Pharmingen,
La Jolla, Calif.) to the cell suspension.
[0140] Cell suspensions were mixed with equal volumes of
fluorochrome labeled monoclonal antibodies (PharMingen), incubated
on ice for 60 minutes and then washed twice with ice cold wash
buffer (PBS, 1% fetal bovine serum, 0.1% sodium azide) prior to
resuspending in 400 ml wash buffer containing 1 mg/ml 7-AAD
(Molecular Probes, Eugene, Oreg.) as a viability marker in some
samples. Flow data was acquired on a FACSCalibur flow cytometer (BD
Immunocytometry Systems, San Jose, Calif.). Both acquisition and
analysis were performed using CellQuest software (BD
Immunocytometry Systems).
[0141] The transgenic animals that expressed either the human or
mouse IL-21 at the highest levels had dramatically altered cell
populations in all lymphoid organs analyzed. Changes seen included
complete loss of thymic cellularity, complete absence of CD45R
positive B cells and increased size and cellularity of spleens.
Both spleen and bone marrow had increased numbers of myeloid sized
cells, which was accounted for by increases in both monocytes and
neutrophils. The pan NK cell marker (DX5) was increased in many
populations. Moderate expressing founders had less dramatic but
still significant changes consistent with the phenotype seen in the
high expressers. Mice with the lowest level of expression had
neither a significant increase in myeloid cells nor decrease in B
cells numbers. They did show significant changes in thymocyte
populations with decreases in CD4+CD8+ double positive cells and
increases in both CD4 and CD8 single positive cells.
Example 3
IL-21 Purified Recombinant Human Protein
Dose-Response Study in Normal Mice
A. Summary
[0142] Normal six week old female C57B1/6 (Harlan Sprague Dawley,
Indianapolis, Ind.). mice were treated by intraperitoneal injection
once daily for either four or eight days with one of four dose
levels of purified recombinant human IL-21 (U.S. Pat. No.
6,307,024) at 0.1, 0.5, 5 or 50 .mu.g/mouse/day or with vehicle as
a control. Body weights and body temperatures were monitored daily.
On either day four or day nine, four of the eight mice from each
protein treatment group and five of the ten mice in the vehicle
control group were sacrificed. Blood, bone marrow and tissues were
harvested and analyzed. Potential perturbations in lymphoid tissues
were examined, as well as general physiologic and toxicological
parameters.
[0143] There was no evidence of toxicity of human IL-21 protein at
any of the doses tested. Body weights and temperatures were
unchanged. There were no apparent changes in clinical chemistry
parameters. However, there were consistent findings relating to
increased percentages of myeloid lineage cells in bone marrow,
spleen and peripheral blood in mice treated with the highest dose
of IL-21 compared to the vehicle control. There was a statistically
significant increase in myeloid lineage sized cells identified by
flow cytometric analysis of spleen homogenate in the high-dose
group. The spleens of the two highest dose groups were
statistically significantly larger than the other groups. On
histopathologic examination, however, only a marginal increase in
extramedullary hematopoiesis was seen in the highest dose group.
There was a statistically significant increase in the myeloid to
erythroid ratio of the bone marrow in the highest dose group
compared to the other groups. Finally, there were increases seen in
peripheral blood both in total white blood cell counts and in the
percentage of monocytes in the same group.
B. Dosing Solution Preparation
[0144] Purified recombinant human IL-21 (U.S. Pat. No. 6,307,024)
was diluted into sterile phosphate buffered saline (GibcoBRL, Grand
Island, N.Y.) at concentrations to deliver 50, 5, 0.5 or 0.1
micrograms of protein in 0.1 ml of PBS vehicle. The doses for the
first four days were made on day 0 and frozen in a frosty
-20.degree. C. freezer prior to use. The doses for days five
through eight were made on day five and frozen as above. Aliquots
of the same PBS were similarly frozen for the vehicle treated
control group. On the day of administration the appropriate
aliquots were thawed and 0.1 ml of solution was injected
intraperitoneally into the mice each day for either four or eight
days.
Study Design
[0145] The mice were six weeks old at the start of the study. Each
treatment group consisted of eight mice, except for the vehicle
control group that included ten mice. One half of the mice in each
treatment group were sacrificed after four days of treatment and
the other half after eight days.
[0146] Before treatment each day, each mouse was weighed and her
body temperature recorded using the Portable Programmable Notebook
System (BMDS, Inc, Maywood, N.J.), by scanning the mouse for
identification number and body temperature from transponders
implanted subcutaneously (IPTT-100, BMDS, Maywood, N.J.).
[0147] At sacrifice, tissues harvested to assess white blood cell
populations by flow cytometric analysis included bone marrow,
thymus and spleen. FACS analysis of the lymphoid organs and bone
marrow was performed with the FACSCalibur, (Becton Dickinson,
Mansfield, Mass.). The tissues harvested for histologic examination
for signs of toxicity of the protein included: spleen, thymus,
liver, kidney, adrenal gland, heart and lungs. All tissues fixed
for histology were kept at 4.degree. C. overnight in 10% Normal
Buffered Saline (NBF) (Surgipath, Richmond, Ill.). The following
day the NBF was replaced with 70% ethanol and the tissues returned
to 4.degree. C. until processing for histology.
[0148] The tissues were processed and stained for Hematoxylin and
Eosin in house, then sent to a contract pathologist for
histopathologic analysis. Blood was collected for complete blood
cell counts (CBC) and serum chemistry profiles. The CBC's were
analyzed in-house with the Cell Dyn 3500 Hematology Analyzer
(Abbott Diagnostics Division, Abbott Park, Ill.) and manual
differential white blood cell counts were analyzed at Phoenix
Central Laboratory, (Everett, Wash.). The serum was kept frozen at
-20.degree. C. until submission to Phoenix Central Laboratory for
complete serum chemistry panels. To assess myeloid:erythroid
ratios, the bone marrow from one femur was applied to CytoSpin
slides (CYTOSPIN 3 CYTOCENTRIFUGE and CYTO SLIDES, Shandon,
Pittsburgh, Pa.) and sent to Phoenix Central Laboratories for
analysis.
Study Results
[0149] There were no apparent clinical indications of physiologic
effects or of toxicity of human IL-21 at doses of 50 .mu.g/day or
lower. Body weights and temperatures remained normal for the
duration of the treatments. Serum chemistry parameters were in
normal ranges. Red blood cell and platelet counts appeared normal.
In the mice receiving 50 .mu.g/day for 8 days, manual differential
white blood cell counts showed that the percentage of monocytes was
elevated in the peripheral blood, and an apparent increase in the
total white blood cell counts. In bone marrow flushed from a femur,
myeloid to erythroid ratios were increased in the 50 .mu.g dose
group, and to a lesser degree the 5 .mu.g dose group from the 8-day
dose set. In a non-parametric multiple column comparison using
InStat (InStat MAC; GraphPad Software, Inc., San Diego, Calif.),
this difference was statistically significant (p=.0049). The
difference between the highest dose group and vehicle was also
significant, (p=.0286). The increased white blood cells in
peripheral blood and the significant increase in myeloid precursors
in the marrow may thus be related.
[0150] Histologic evaluation of the following tissues showed no
apparent evidence of cytologic or structural changes, mitotic
events or necrosis: thymus, liver, kidney, adrenal gland, duodenum,
pancreas, jejunum, caecum, colon, mesenteric lymph nodes, uterus,
ovary, salivary gland, heart, trachea, lung, and brain. There were
no apparent differences between the treatment groups in the weights
of the thymus, kidney, liver or brain. Of all the tissues examined,
only the spleen weights were significantly affected.
[0151] Each mouse spleen weight was normalized to her brain weight.
In the 50 .mu.g/day treatment group compared to the vehicle, 0.1
.mu.g and 0.5 .mu.g treatment groups, the average of the spleen
weights was nearly 50% greater after four days of treatment and
almost 100% greater after eight days than the average spleen
weights of the other three groups. In the four-day set, the 5
.mu.g/day group also tended to have larger spleens than the control
and low dose groups. The difference in the spleen/brain weights
with data from the four-day and the eight-day sets combined by
treatment group was statistically significant (p=.0072) by
Kruskall-Wallace non-parametric ANOVA, multiple column comparison
test using the InStat program (GraphPad Software).
[0152] A marginal increase in extrameduallary hematopoiesis,
especially in the red pulp was seen in spleens of mice from the
highest dose group, even in the mice treated for four days. Flow
cytometric analysis of the spleens showed a significant increase in
the proportion of myeloid size cells in the highest dose group
(p=0.01, Student's t test), representing increases in both
monocytes and neutrophils. This effect may be related to the
increased peripheral blood mononuclear cell percentage, as well as
the apparent increase in myeloid precursors in the bone marrow,
described above. Moreover, the transgenic mice derived from
insertion of the human zalpha 11 gene had increased extramedullary
hematopoiesis in their spleens compared to non-transgenic litter
mates.
[0153] Several changes were observed in the 50 .mu.g per day dose
group compared to the control group that implicate IL-21 in
production or development of cells of the myeloid lineage. Taken
together, the observed changes suggest that zalpha 11 may be useful
as a therapeutic protein in such medical specialties as cancer and
immunologic disorders described herein.
Example 4
Preliminary Elimination and Tissue Distribution Study of Purified
Recombinant Human IL-21 Protein
A. Summary
[0154] In order to elucidate tissue distribution and elimination
patterns of the purified rhIL-21, a preliminary pharmacokinetic
study was undertaken. Nine week old male C57B1/6 mice were given
purified recombinant human IL-21 protein labeled with
.sup.111Indium (.sup.111In) (NEN, Boston, Mass.) by one of three
routes. A single bolus injection was given to each mouse by either
the intravenous (IV), intraperitoneal (IP), or subcutaneous route
(SC). The mice injected by either the subcutaneous or
intraperitoneal route were sacrificed at either one or three hours
after injection. The mice injected intravenously were sacrificed
after either ten minutes or one hour following injection. Blood,
plasma and selected tissues were harvested at various timepoints
and counted by a gamma counter to estimate the approximate
half-life and tissue distribution of the exogenous labeled protein.
The tissues that were harvested for counting as well as the
intervals of sacrifice were selected based on reports of the
distribution of other cytokines labeled with radionuclides.
[0155] At sacrifice, tissues harvested for counting of
radioactivity included thymus, spleen, kidney, a lobe of liver, a
lobe of lung, and urinary bladder. In the group receiving the
injection intraperitoneally, gut was also counted to assess
incidence of injection into the gut, and in the subcutaneously
dosed mice, skin with underlying structures in the area of
injection was counted. The cpm for whole liver and lung were
calculated from a section that was counted and a percentage of the
whole organ weight represented by the section.
[0156] After the end of the study the collected tissues, whole
blood and plasma were counted on the COBRA II AUTO-GAMMA.RTM. gamma
counter (Packard Instrument Company, Meriden, Conn.). An aliquot of
the original labeled dosing solution was also counted at the end of
the study with the tissues. This allowed calculation of percent
total injected radioactivity for each mouse and simultaneous
correction of all counts for radioactive decay. Approximations of
remaining blood volume and organ weights indicated that the
majority of the counts administered were accounted for, and
therefore the percentage of counts per tissue were a reasonable
representation of distribution of the counts following labeled
IL-21 administration by each route.
B. .sup.111Indium Labeling of IL-21
[0157] Purified recombinant human IL-21 (U.S. Pat. No. 6,307,024)
was conjugated with a 10 fold molar excess of DTPA (Peirce,
Rockford, Ill.) by incubating 30 minutes at room temperature in
PBS. Unreacted DTPA and hydrolyzates were removed by buffer
exchange on a Biomax-5k NMWL (Ultrafree-15, Millipore, Bedford,
Mass.). The void volume protein peak was concentrated to 5 mg/ml
and an aliquot taken for testing in a bioassay (anti-CD40
stimulation of murine B-cells (Example 10)). Upon confirming that
the DTPA-conjugate still had full bioactivity the conjugate was
diluted to 0.5 mg/ml with 1M Na Acetate pH 6.0. Two mCi of
.sup.111Indium was taken up in 0.5 ml 1M Na Acetate pH 6.0 and
mixed with the DTPA-human IL-21 for 30 min. at room temperature.
Unincorporated .sup.111Indium was removed during buffer exchange to
PBS on a PD-10 column (Pharmacia, Piscataway, N.J.). The
radio-labeled material was diluted with unlabeled human IL-21 to
give a specific activity of 100 mCi/mg, sterile filtered and stored
at 4.degree. C. overnight. One hundred percent of the labeled
protein was retained on a Biomax-5k NMWL membrane (Millipore). The
labeled .sup.111In-human IL-21 was administered to mice in the
elimination and pharmacokinetic studies. Fifty .mu.g human IL-21
protein labeled with 5 .mu.Ci of labeled human IL-21 in 0.1 ml of
PBS vehicle was administered to each animal.
C. Results Of Preliminary Distribution Study
[0158] After one and three hours following administration by all
three routes, the highest concentration of .sup.111In-human IL-21,
was found in kidney and the second highest was in urine and urinary
bladder, as evinced by these tissues having the highest cpm. The
average counts recovered from kidneys were from 3 to 8 times higher
than the whole liver counts, depending on the route of injection
and the sacrifice timepoint. For example, the average kidney cpm at
60 minutes following IV injection was 4.5 times greater than the
average counts calculated for whole liver from the same group. In
the group that was sacrificed ten minutes after intravenous
administration, the highest cpm was again in kidney, and the second
highest accumulation was equivalent in liver, urinary bladder and
urine.
D. Preliminary Pharmacokinetic Study
[0159] Blood and plasma collections were done at 10, 30 and 60
minutes following injection by all three routes. Following
injection by the IV route, a separate set of mice had blood and
plasma samples taken at two, five and ten minutes. Another set of
mice who received their injections by either the IP or SC route had
blood sampled at one, two and three hours. For the treatment groups
see Table 4. The short collection times bracket the reported
half-life of IL-2 following intravenous injection. The reported
T1/2 was in the range of 2.5 to 5.1 minutes. For reference to in
vivo administration to IL-2, see Donohue J H and Rosenberg S A J
Immunol 130:2203, 1983. The long timepoints were chosen to outline
the anticipated elimination phase. TABLE-US-00004 TABLE 4 Route of
injection Bleed Times (min.) Sacrifice Time Intravenous Group 1 2,
5, 10 10 min. Intravenous Group 2 10, 30, 60 60 min.
Intraperitoneal Group 1 10, 30, 60 60 min. Intraperitoneal Group 2
60, 120, 180 180 min. Subcutaneous Group 1 10, 30, 60 60 min.
Subcutaneous Group 2 60, 120, 180 180 min.
[0160] Un-labeled IL-2 has been shown to be eliminated from the
serum with a half-life of approximately three minutes in mice after
IV injection. For reference see Donahue, J H and Rosenburg supra.
Following IP and SC injection of similar amounts of IL-2, the
duration of persistence of IL-2 activity in serum was prolonged
from 2 units/ml for less than 30 minutes following IV injection to
greater than 2 units/ml for 2 hours following IP and 6 hours
following SC injections. The principle route of clearance of IL-2
appears to be the kidney. IL-21 has been shown to be structurally
similar to IL-2, as discussed herein. Preliminary evaluation of the
elimination of IL-21 appears to be consistent with the apparent
clearance of IL-2 by the kidneys, based on the accumulation of cpm
predominantly in the kidneys, followed by the urinary bladder and
urine in the present study.
[0161] Estimations were made of pharmacokinetic parameters based on
non compartmental analysis of the cpm data obtained from the
plasma, using the PK analysis program WinNonLin, Version 1.1,
(Scientific Consulting Inc., Cary, N.C.). Plasma half-lives of
IL-21 were estimated using the predicted terminal elimination rate
constants for intravenous, subcutaneous, and intraperitoneal
administration of a 50 .mu.g dose. The pharmacokinetic results were
estimations due to limited data points in the terminal elimination
region of the plasma concentration vs. time profiles. Moreover, the
fit of the terminal elimination phase for SC and IP dosing required
use of data from timepoints during which absorption of the
.sup.111In-human IL-21 was apparently still occurring. However,
estimations of half-lives following intravenous, subcutaneous, and
intraperitoneal dosing were 13.6 min., 18.8 min., and 34.3 min.,
respectively. Since a dosing range was not evaluated it was not
apparent whether saturable or active elimination (Michaelis Menten
kinetics) was occurring. Therefore, these half-life calculations
are estimations.
[0162] Estimates of the bioavailability of the labeled protein were
made based on the area under the curve (AUC) following subcutaneous
or intraperitoneal dosing compared to that of intravenous dosing.
The estimated bioavailability following subcutaneous and
intraperitoneal injection were 35.8% and 63.9% respectively.
Because only one protein dose was studied, the bioavailability was
not evaluated as a function of dose. The estimated clearance and
volume of distribution (based on the data from the intravenous
injection) were 0.48 ml/min. and 6.1 ml, respectively.
[0163] Although the data are preliminary, the fate of IL-21
administered IV was similar to that reported for IL-2, another
4-helix bundle cytokine (Donahue, J H and Rosenburg, S A supra.).
Like IL-2, IV-administered IL-21 had a plasma half life of only
minutes with the main clearance in the kidney. Three hours after
injection, the majority of the labeled material extracted from
kidney was still retained in a Biomax 5K NMLW membrane (Millipore).
Since it has previously been reported that the indium remains
associated with protein even during lysosomal degradation (Staud,
F. et al., J. Pharm. Sciences 88:577-585, 1999) IL-21 is
accumulating and may be degraded in the kidney. The current study
also showed, as observed with many other proteins, including IL-2
(Donahue, J H and Rosenburg, S A, supra.), that IP and SC
administration significantly prolonged the plasma levels of
IL-21.
Example 5
Isolation and Expansion of Fresh Human Bone Marrow MNC CD34+
Fraction Using IL-21 for Assessment of NK Activity
A. Selection and Isolation of CD34+ cells from human Bone
Marrow
[0164] Fresh human bone marrow mononuclear cells (MNC) were
prepared to enrich for cells having NK cell activity. Fresh human
MNCs were obtained from Poeitic Technologies (Gaithersburg, Md.).
10 ml alpha MEM (JRH, Lenexa, Kans.) containing 10% HIA FBS
(Hyclone, Logan, Utah) and the antibiotic 1% PSN (Gibco, BRL, Grand
Island, N.Y.) was added to the cell suspension and the cells were
passed through a 100 .mu.m sieve. The cells were then counted,
pelleted, washed with 10 ml PBS containing 2% FBS, then pelleted
again and resuspended in 1 ml PBS containing 2% FBS. Cells having a
CD34 cell surface marker (CD34+ cells) were magnetically separated
using a Detachabead kit with Dynabeads M-450 CD34 ((Dynal, Oslo,
Norway), as per manufacturer's instructions. Both the CD34+ cell
and the CD34- cell fractions were further analyzed below.
B. Expansion of CD34+ cells using IL-21
[0165] A CD34+ cell fraction was plated into four wells in a
24-well plate. 50,000 positively selected cells suspended in 1 ml
Alpha MEM (JRH) containing 10% HIA FBS (Hyclone) and 1% PSN
(Gibco/BRL), plus the various cytokines described below were plated
in each of the 4 wells (1-4). Various reagents were used to test
for IL-21-induced expansion of the CD34+ selected bone marrow MNCs:
Reagents included human flt3 (R&D, Minneapolis, Minn.);
purified human IL-21 (U.S. Pat. No. 6,307,024); human IL-15
(R&D). Reagents were combined as follows at day 0: In well #1,
2 ng/ml human flt3 was added. In well #2, 2 ng/ml human flt3 and 15
ng/ml purified human IL-21 were added. In well #3, 2 ng/ml human
flt3 and 20 ng/ml human IL5 were added. In well #4, 2 ng/ml human
flt3, 15 ng/ml purified human IL-21, and 20 ng/ml human IL15 were
added. After incubating for 18 days, the suspension cells from each
well were pelleted, and then resuspended in 0.5 ml alpha MEM (JRH)
containing 10% HIA FBS (Hyclone) and 1% PSN (Gibco/BRL), and
counted to assess proliferation of the CD34+ cell fraction. A low
level of proliferation was seen in the presence of flt3 alone
(control well #1), but the presence of IL-15 or IL-21 in addition
to flt3 had not significant effect on the expansion (wells, #2 and
#3). However, expansion beyond the flt3 control was evident in well
#4 which contained IL-15 and IL-21 in addition to flt3. This result
suggested that IL-21 and IL-15 act in synergy to expand the human
CD34+ cell population. Moreover, the results of this experiment
supported the results seen with the mouse IL-21 in the mouse BM
assay (Example 1).
[0166] All cell populations were then tested for NK activity and
subjected to flow cytometry analysis, as shown below (Example
7).
C. Expansion of CD34+ or CD34- cells using IL-21 with delayed
addition of IL-15
[0167] Both CD34 positive and negative (CD34-) fractions were
plated separately into six 12 well plate wells (1-6). Each of six
wells contained 100,000 positively or negatively selected cells in
2 ml alpha MEM containing 10% HIA FBS and PSN, described above.
Reagents used were as described above. In well #1, 2 ng/ml human
flt3 was added at day 0. In well #2, 2 ng/ml human flt3 was added
at day 0, and after 5 days incubation 20 ng/ml human IL15 was
added. In well #3, 2 ng/ml human flt3 and 15 ng/ml human IL-21 were
added at day 0. In well #4, 2 ng/ml human flt3 and 15 ng/ml human
IL-21 were added at day 0, and after 5 days incubation 20 ng/ml
human IL15 was added. In well #5, 2 ng/ml human flt3 and 20 ng/ml
human IL15 were added at day 0. In well #6, 2 ng/ml human flt3, 15
ng/ml human IL-21, and 20 ng/ml human IL15 were added at day 0.
After incubating for a total of 15 days from the start of the
experiment, the cells from each well were harvested and
counted.
[0168] In the CD34+ population a low level of proliferation was
seen in the presence of flt3 alone (control well #1), but the
presence of IL-15 or IL-21 added at day 0 in addition to flt3 had
no significant effect on the expansion (wells, #3 and #5). Addition
of IL-15 after 5 days had some proliferative effect in comparison
to the flt3 control (well #2 compared to well #1) and a
proliferative effect in the presence of zalpha 11 (well #4 compared
to well #3). However, the greatest expansion was evident in well #6
which contained IL-15 and IL-21 in addition to flt3 at day 0.
[0169] In the CD34- population, no proliferation was seen in the
presence of flt3 alone (control well #1), and in fact a decrease in
the cell population was evident. The presence of zalpha 11 added at
day 0 in addition to flt3 (well #3) was similar to the flt3
control. The presence of IL-15 added at day 5 increased
proliferation effect of the cells in the presence (well #4) or
absence (well #2) of IL-21. Again, the greatest expansion was
evident in well #6 which contained IL-15 and IL-21 in addition to
flt3 at day 0.
[0170] All cell populations were then tested for NK activity and
subjected to FACS analysis, as shown below (Example 7).
Example 6
Isolation and Expansion of Fresh Mouse Cells Using Human and Mouse
IL-21 for Assessment of NK Activity and NK Cell Markers
A. Isolation and Expansion of Fresh Mouse Low Density Bone Marrow
Cells Using Human and Mouse IL-21
[0171] Fresh mouse marrow cells were isolated by clipping both ends
of mouse femurs, and flushing two to three milliliters of growth
medium (see below) through the inside of the bone into a collection
tube. The growth medium was 500 ml RPMI 1640 Medium (JRH
Biosciences. Lenexa, Kans.); 5 ml 100.times. L-glutamine (Gibco
BRL. Grand Island, N.Y.); 5 ml 100.times. Na Pyruvate (Gibco BRL);
5 ml 100.times. Penicillin, Streptomycin, Neomycin (PSN) (Gibco
BRL); and 50 ml heat-inactivated Fetal Bovine Serum (FBS) (Hyclone
Laboratories. Logan, Utah). The marrow cells were then broken-up by
pipeting the media up and down several times. The cells were then
pelleted and washed once with growth medium, and passed through a
70-micron sieve. The low-density mononuclear cells were then
isolated by subjecting the marrow cells to a density gradient.
Marrow cells in five to eight milliliters of growth medium were
carefully pipetted on top of five to eight milliliters of NycoPrep
1.077 Animal (Nycomed. Oslo, Norway) in a centrifuge tube. This
gradient was then centrifuged at 600.times. g for 20 minutes. The
low density mononuclear cells were harvested from the interface
layer between the NycoPrep and the medium. These cells were then
diluted to approximately 20 milliliters in growth medium, pelleted
and washed. The cells were then plated at approximately
0.5-1.5.times.10.sup.6 cells per milliliter in growth medium in a
standard tissue culture flask and incubated at 37.degree. C., 5%
CO.sub.2 for two hours.
[0172] The non-adherent, low density (NA LD) marrow cells were then
harvested and plated at 0.5-2.0.times.10.sup.5 cells per milliliter
in growth medium plus 2.5 nanograms per milliliter mouse flt3 (R
and D Systems. Minneapolis, Minn.) plus 25 to 50 nanograms per
milliliter human Interleukin 15 (IL-15) (R and D Systems) with or
without 50 to 150 nanograms per milliliter human IL-21; or with or
without 0.12 to 10 nanograms per milliliter mouse IL-21.
[0173] There was no significant expansion without the addition of
the human or mouse IL-21. Non-adherent cells were expanded in the
cultures containing mouse IL-21 as low as 0.12 ng/ml and in the
cultures containing human IL-21 as low as 22 ng/ml. In cultures
containing both the human and mouse IL-21, non-adherent cell
expansion increased with increasing dose if IL-21, with the mouse
ligand saturating response at about 5-10 ng/ml and the human not
reaching a saturating response even at the highest dose of 200
ng/ml. Human IL-21 appeared to be approximately 20 to 100 fold less
potent on mouse cells as the mouse IL-21. After approximately five
to ten days the IL-21 expanded mouse cells were harvested and
analyzed by flow cytometry (FACSCalibur; Becton Dickinson,
Mansfield, Mass.) to determine what percentage of them were
positive for NK cell antigens, where 46% were positive for the
PanNK cell marker DX5 (Pharmingen).
B. Isolation and Expansion of Fresh lineage Depleted Mouse Marrow
Cells
[0174] Fresh mouse lineage depleted (lin-) marrow cells were
isolated from fresh mouse marrow cells by first incubating the
cells with the following antibodies: TER119, Gr-1, B220, MAC-1,
CD3e and I-Ab (Pharmingen. San Diego, Calif.). The lin+ cells were
then removed with Dynabeads M-450 sheep anti-rat IgG (Dynal, Lake
Success, N.Y.) as per manufacturer's instructions.
[0175] The negatively selected lin- marrow cells were then plated
as above in growth medium plus either 2.5 ng/mL flt3 (R&D
Systems) and 25 ng/mL IL-15 (R&D Systems); or flt3, IL-15 and
mouse IL-21, 2 to 5% BHK mouse IL-21 conditioned medium. After six
days of growth, the cultures were harvested, counted and submitted
to an NK cell activity assay (Example 7). Cells grown with mouse
IL-21 were approximately two to three times more effective at
lysing NK cell target cells (YAC-1 cells) as the cells grown
without IL-21.
C. Isolation and Expansion of CD4- CD8- (Double Negative or DN)
Thymocytes
[0176] Fresh mouse thymocytes were isolated by chopping and sieving
thymuses from three to eight week old mice. CD4- CD8- (DN) cells
were then negatively selected by incubating the thymocytes with
anti-CD4 and anti-CD8 antibodies (PharMingen), then removing the
CD4+ CD8+ cells with Dynabeads M-450 sheep anti-rat IgG (Dynal) as
per manufacturer's instructions.
[0177] The DN mouse thymocytes were then grown in growth medium
plus 2.5 ng/mL flt3 (R&D Systems), 25 ng/mL IL-15 (R&D
Systems) and 10 ng/mL IL-7 (R&D Systems) with or without mouse
IL-21 as above. Six days later the cells were harvested, counted,
analyzed by flow cytometry as described above, and also submitted
to an NK cell activity assay (Example 7).
[0178] The culture grown with mouse IL-21 yielded approximately
480,000 cells while the culture without IL-21 yielded only
approximately 160,000 cells. The culture grown with mouse IL-21 was
found to be approximately 16.2% positive for the NK cell antigen
Pan NK, DX5 (PharMingen). The culture grown without IL-21 was 14.6%
positive for DX5. The cells grown with IL-21 lysed NK cell target
cells, YAC-1, approximately two times better than the cells grown
without IL-21. The expanded cells did not lyse significantly a
negative control target cell line, EL4. These results suggested
that IL-21 selectively expands lytic NK cells.
Example 7
Activity of Human and Mouse IL-21 Expanded Cells and Mature Murine
NK Cells in NK
Cell Cytotoxicity Assays
A. NK Cell Assay
[0179] NK cell-mediated target cytolysis was examined by a standard
.sup.51Cr-release assay. Target cells (K562 cells (ATCC No.
CCL-243) in human assays, and YAC-1 cells (ATCC No. TIB-160) in
mouse assays) lack expression of major histocompatability complex
(MHC) molecules, rendering them susceptible to NK cell-mediated
lysis. A negative control target cell line in mouse assays is the
MHC.sup.+ thymoma EL4 (ATCC No. TIB-39). We grew K562, EL4, and
YAC-1 cells in RP10 medium (standard RPMI 1640 (Gibco/BRL, Grand
Island, N.Y.) supplemented with 10% FBS (Hyclone, Logan, Utah), as
well as 4 mM glutamine (Gibco/BRL), 100 I.U./ml penicillin+100
MCG/ml streptomycin (Gibco/BRL), 50 .mu.M .beta.-mercaptoethanol
(Gibco/BRL) and 10 mM HEPES buffer (Gibco/BRL). On the day of
assay, 1-2.times.10.sup.6 target cells were harvested and
resuspended at 2.5-5.times.10.sup.6 cells/ml in RP10 medium. We
added 50-100 .mu.l of 5 mCi/ml .sup.51Cr-sodium chromate (NEN,
Boston, Mass.) directly to the cells and incubated them for 1 hour
at 37.degree. C., then washed them twice with 12 ml of PBS and
resuspended them in 2 ml of RP10 medium. After counting the cells
on a hemacytometer, the target cells were diluted to
0.5-1.times.10.sup.5 cells/ml and 100 .mu.l (0.5-1.times.10.sup.4
cells) were mixed with effector cells as described below.
[0180] In human assays, effector cells were prepared from selected
and expanded human CD34.sup.+ BM cells (Example 5B) which were
harvested, washed, counted, mixed at various concentrations with
.sup.51Cr-labeled target cells in 96-well round bottomed plates,
and incubated for 4 hours at 37.degree. C. After co-incubation of
effector cells and the labeled target cells, half of the
supernatant from each well was collected and counted in a gamma
counter for 1 min/sample. The percentage of specific .sup.51Cr
release was calculated from the formula 100.times.(X-Y)/(Z-Y),
where X is .sup.51Cr release in the presence of effector cells, Y
is the spontaneous release in the absence of effectors, and Z is
the total .sup.51Cr release from target cells incubated with 0.5%
Triton X-100. Data were plotted as the % specific lysis versus the
effector-to-target ratio in each well.
B. Activity of Human IL-21 Expanded Cells
[0181] Isolated CD34.sup.+ human HPCs cultured with flt3+/-IL-21
and flt3+IL-15+/-IL-21 ( Example 5), were harvested the cells on
day 15 to assess their capacity to lyse MHC-K562 cells in a
standard .sup.51Cr-release assay as described above, and to analyze
their surface phenotype by flow cytometry. As expected from
previous reports (Mrozek, E et al., Blood 87:2632-2640, 1996; and
Yu, H et al., Blood 92:3647-3657, 1998), simultaneous addition of
IL-15 and flt3L did induce the outgrowth of a small population of
CD56.sup.+ cells. Interestingly, although BM cells cultured
simultaneously with IL-21 and flt3L did not expand significantly,
there was a significant increase in total cell numbers in cultures
containing a combination of flt3L, IL-21 and IL-15 (see, Example
5).
[0182] For an assessment of the surface phenotype of these human BM
cultures, we stained small aliquots of the cells for 3-color flow
cytometric analysis with anti-CD3-FITC, anti-CD56-PE and
anti-CD16-CyChrome mAbs (all from PharMingen, San Diego, Calif.)
and analyzed them on a FACSCalibur using CellQuest software (Becton
Dickinson, Mountain View, Calif.). This flow cytometric analysis
confirmed that the cells growing out of these cultures were
differentiated NK cells, as they were large and granular and
expressed both CD56 and CD16, and were CD3.sup.- (Lanier, L L Annu.
Rev. Immunol. 16:359-393, 1998). Furthermore, these cells exhibited
significantly higher effector function than those cells grown with
IL-15 and flt3. More specifically, cells grown in all three
cytokines lysed more than 40% of the K562 targets at an
effector-to-target ratio (E:T) of 1.5, whereas cells grown in
IL-15+flt3L lysed fewer than 5% of the targets at an E:T of 2.
These data demonstrate that, in combination with IL-15, IL-21
stimulates the differentiation of NK cells from CD34.sup.+ BM
cells.
C. Activity of Mouse IL-21 Expanded Cells
[0183] To test the effects of IL-21 on murine hematopoietic
progenitor cells, purified Lineage-negative (Lin-) bone marrow
cells from C57B1/6 mice were expanded in flt3+IL-15+/-IL-21, as
described in Example 6B. On day 6 of culture, the cells
("effectors") were harvested and counted, then resuspended in 0.4
ml of RP10 medium (Example 7A). Two aliquots (0.15 ml each) of each
sample expanded with or without IL-21 (Example 7A) were diluted
serially 3-fold in duplicate in 96-well round bottomed plates, for
a total of 6 wells of 100 .mu.l each. The remaining 100 .mu.l of
cells were stained for NK cell surface markers with FITC-anti-2B4
and PE-anti-DX5 mAbs (PharMingen) and analyzed by flow cytometry.
Each group of cells exposed to flt3+IL-15 with or without the
presence of IL-21 had similar fractions of 2B4+DX5+cells, ranging
from 65-75% positive for both NK markers.
[0184] For the NK lysis assay, target cells (YAC-1 and EL4) were
labeled with .sup.51Cr as described above. After counting the
target cells on a hemacytometer, the target cells were diluted to
0.5-1.times.10.sup.5 cells/ml and 100 .mu.l of YAC-1 or EL4
(0.5-1.times.10.sup.4 cells) were mixed with 100 .mu.l effector
cells and incubated for 4 hours at 37.degree. C. Specific lysis was
determined for each well as described above.
[0185] We found that cells grown in the presence of
flt3+IL-15+IL-21 exhibited enhanced lytic activity (roughly 2-fold)
against the YAC-1 targets (but did not kill the MHC+control cell
line EL4). At an effector-to-target ratio (E:T) of 5, NK cells
generated in the presence of all 3 cytokines (IL-21+flt3+IL-15)
lysed 12% of the YAC-1 cells, whereas those NK cells expanded with
flt3+IL-15 lysed 6% of the YAC-1 targets. Subsequent experiments
confirmed this trend.
[0186] In a second approach to determine the biological activity of
IL-21 on murine NK cells, we isolated immature CD4.sup.-CD8.sup.-
("double negative", DN) mouse thymocytes as described in Example 6C
and cultured them with IL-15+flt3+IL-7 or IL-15+flt3+IL-2, with or
without IL-21. On day 6 of culture, the cells were harvested and
assayed for NK lytic activity on YAC-1 and EL4 cells as described
above. We found that cells cultured in the presence of IL-21 had
the greatest lytic activity in this assay, with enhanced lytic
activity over those cells cultured in the presence of the other
cytokines. Specifically, DN thymocytes grown with IL-15+flt3+IL-7
killed 18% of the YAC-1 cells at E:T of 24 while cells grown in the
presence of IL- 15+flt3+IL-7 plus IL-21 killed 48% of the targets
at the same E:T. DN thymocytes grown in IL-15+flt3+IL-2 killed 15%
of the YAC-1 targets at an E:T of 6, whereas cells grown with these
3 cytokines and IL-21 killed 35% of the YAC-1 cells at an E:T of 9.
Flow cytometry was performed on the cultured cells one day before
the NK lysis assay. As was true for the bone marrow cultures,
despite the proliferative effect of IL-21 (cell numbers increase
approximately 2-fold when IL-21 is added), it did not significantly
enhance the fraction of DX5.sup.+ cells (17-20% of total cells in
the cultures with IL-7, and 35-46% of total in cultures with IL-2).
These data imply that IL-21, in combination with IL-15 and flt3,
enhances the lytic activity of NK cells generated from murine bone
marrow or thymus.
D. Activity of Mouse IL-21 on Mature Murine NK Cells
[0187] In order to test the effects of mouse IL-21 on mature NK
cells, we isolated spleens from four 5-week old C57B1/6 mice
(Jackson Laboratories, Bar Harbor, Me.) and mashed them with
frosted-end glass slides to create a cell suspension. Red blood
cells were removed by hypotonic lysis as follows: cells were
pelleted and the supernatant removed by aspiration. We disrupted
the pellet with gentle vortexing, then added 900 .mu.l of sterile
water while shaking, followed quickly (less than 5 sec later) by
100 .mu.l of 10X HBSS (Gibco/BRL). The cells were then resuspended
in 10 ml of 1X HBSS and debris was removed by passing the cells
over a nylon mesh-lined cell strainer (Falcon). These RBC-depleted
spleen cells were then pelleted and resuspended in MACS buffer
(PBS+1%BSA+2 mM EDTA) and counted. We stained 300.times.10.sup.6 of
the cells with anti-DX5-coated magnetic beads (Miltenyi Biotec) and
positively selected DX5.sup.+ NK cells over a MACS VS+ separation
column, according to the manufacturer's instructions, leading to
the recovery of 8.4.times.10.sup.6 DX5.sup.+ cells and
251.times.10.sup.6 DX5.sup.- cells. Each of these groups of cells
were cultured in 24-well plates (0.67.times.10.sup.6 cells/well, 2
wells per treatment condition) in RP10 medium (Example 7A) alone or
with 1)30 ng/ml mouse IL-21, 2) 30 ng/ml recombinant mouse IL-2
(R&D Systems, Inc., Minneapolis, Minn.), 3) 30 ng/ml
recombinant human IL-15 (R&D), 4)30 ng/ml each of mouse IL-21
and hIL-15, or 5)30 ng/ml each of mIL-2 and hIL-15. The cells were
harvested after 21 hours, washed, and resuspended in RP10 medium
and counted. The cells were then assayed for their ability to lyse
.sup.51Cr-labeled YAC-1 or EL4 targets cells, as described in
Example 7A.
[0188] In general, there was little NK activity from the DX5.sup.-
(non-NK cells) groups, but the DX5.sup.- cells cultured with IL-21
and hIL-15 did lyse 25% of the YAC-1 target cells at an E:T of 82.
By comparison, DX5.sup.- cells cultured with hIL-15 alone lysed 14%
of the YAC-1 targets at an E:T of 110. This suggests that IL-21 and
IL-15 are acting together on the residual NK1.1.sup.+ NK cells in
this cell preparation. As for the DX5.sup.+ cell preparation,
treatment with mouse IL-21 alone did not significantly increase
their effector function (their lysis of YAC-1 cells was similar to
the untreated group). As expected, both IL-2 and IL-15
significantly improved NK activity. The highest level of lysis,
however, was detected in the group treated with IL-21 and hIL-15
(65% lysis of YAC-1 cells at an E:T of 3.3, vs. 45% lysis at an E:T
of 4 for the hIL- 15 treatment group). Taken together, these
results suggest that although IL-21 alone may not increase NK cell
lysis activity, it does enhance NK lysis activity of mature NK
cells, when administered with IL-15.
Example 8
IL-21 Proliferation of Human and Mouse T-cells in a T-cell
Proliferation Assay
A. Murine IL-21 Proliferation of Mouse T-cells
[0189] T cells from C57B1/6 mice (Jackson Laboratories, Bar Harbor,
Me.) were isolated from pooled splenocytes and lymphocytes from
axillary, brachial, inguinal, cervical, and mesenteric lymph nodes
(LNs). Spleens were mashed with frosted-end glass slides to create
a cell suspension. LNs were teased apart with forceps and passed
through a cell strainer to remove debris. Pooled splenocytes and LN
cells were separated into CD8.sup.+ and CD4.sup.+ subsets using two
successive MACS magnetic separation columns, according to the
manufacturer's instructions (Miltenyi Biotec, Auburn, Calif.).
Whole thymocytes were collected from the same mice.
[0190] Cells were cultured at 3.times.10.sup.5 cells/well
(thymocytes) or 10.sup.5 cells/well (mature T cells) with
increasing concentrations of purified murine IL-21 (0-30 ng/ml)
(U.S. Pat. No. 6,307,024) in 96-well flat bottomed plates
pre-coated overnight at 4.degree. C. with various concentrations of
anti-CD3 mAb 2C11 (PharMingen) for 3 days at 37.degree. C. The
anti-CD3 antibody served to activate the murine T-cells through the
T-cell receptor. Each well was pulsed with 1 .mu.Ci
.sup.3H-thymidine on day 2 and plates were harvested and counted 16
hours later to assess proliferation.
[0191] When we tested IL-21 in T cell proliferation assays, we
found that it co-stimulated anti-CD3-activated murine thymocytes,
leading to an accelerated outgrowth of CD8.sup.+CD4.sup.- cells
(the majority of the thymocytes cultured with anti-CD3+IL-21 were
CD8+CD4 by day 3 of culture, while cells cultured with anti-CD3
alone did not significantly skew to this phenotype until day 5). We
did not observe significant levels of proliferation of thymocytes
to IL-21 in the absence of anti-CD3.
[0192] Interestingly, when we assayed mature peripheral murine T
cells for their ability to respond to IL-21+anti-CD3, we found that
only the CD8.sup.+, but not the CD4.sup.+ subset, responded in a
dose-dependent manner to IL-21. We also observed weak but
reproducible proliferation of CD8.sup.+ cells (but not CD4.sup.+
cells) in response to IL-21 alone. Interestingly, this was not
observed for human T cells (see Example 8B, below).
B. Human IL-21 Proliferation of Human T-cells
[0193] Human CD4+ and CD8+ T cells were isolated from PBMC as
described in Example 9 (below) Cells were cultured at about
10.sup.5 cells/well with increasing concentrations of purified
human IL-21 (0-50 ng/ml) (U.S. Pat. No. 6,307,024) in 96-well flat
bottomed plates pre-coated overnight at 4.degree. C. with various
concentrations of anti-human CD3 mAb UCHT1 (PharMingen) for 3 days
at 37.degree. C. Each well was pulsed with luCi .sup.3H-thymidine
on day 2 and plates were harvested and counted 16 hours later.
Unlike our results with mouse T cells, our preliminary data
suggests that human IL-21 co-stimulates CD4+, but not CD8+, human T
cells in a dose-dependent fashion.
[0194] In other experiments, mature murine CD4+ and CD8+ T cells
were enriched from pooled C57B1/6 spleen and LN cells by depletion
of CD19.sup.+ B cells using a magnetic bead column. The resulting
cell populations were assayed for proliferation to plate-bound
anti-mouse CD3.epsilon. mAb in the absence or presence of
increasing concentrations of murine IL-21, as indicated. Data shown
are representative of results from 4 experiments.
[0195] T cells from C57B1/6 mice were isolated from pooled
splenocytes and lymphocytes from auxiliary, brachial, inguinal,
cervical, and mesenteric LNs. Spleens were mashed with frosted-end
glass slides to create a cell suspension. LNs were teased apart
with forceps and passed through a cell strainer to remove debris.
Pooled splenocytes and LN cells were separated into CD8.sup.+ and
CD4.sup.+ subsets using two successive MACS magnetic separation
columns, according to the manufacturer's instructions (Miltenyi
Biotec, Sunnyvale, Calif.). Cells were cultured at 10.sup.5/well
with increasing concentrations of murine IL-21 (0-30 ng/ml) in
96-well flat bottomed plates pre-coated overnight at 4.degree. C.
with various concentrations of anti-CD3.epsilon. mAb 2C11
(PharMingen) for 3 days at 37.degree. C. Each well was pulsed with
1 .mu.Ci .sup.3H-thymidine on day 2 and plates were harvested and
counted 16 hours later.
[0196] Table 5 illustrates that mIL-21 co-stimulates the
proliferation of murine CD8+ T cells. Values represent the CPM
incorporated of .sup.3H-thymidine (average +/-standard deviation of
triplicate wells). TABLE-US-00005 TABLE 5 Anti-CD3 mAb (ug/ml)
ng/ml mIL-21 0 0.11 0.33 1.0 3.0 CD4+ 0 405 +/- 101 67895 +/- 18752
141175 +/- 6733 202251 +/- 35571 246626 +/- 45106 1.2 247 +/- 86
80872 +/- 23598 126487 +/- 7472 178863 +/- 33583 205861 +/- 14675 6
302 +/- 106 75192 +/- 5323 102005 +/- 20059 191598 +/- 15881 218718
+/- 12142 30 364 +/- 126 86164 +/- 8065 141065 +/- 4921 186089 +/-
17585 266650 +/- 39839 CD8+ 0 168 +/- 47 40198 +/- 4557 70272 +/-
4141 84771 +/- 9450 97869 +/- 3368 1.2 268 +/- 117 50095 +/- 3959
84319 +/- 6373 105176 +/- 10828 113394 +/- 3657 6 323 +/- 159 78113
+/- 6967 108461 +/- 2175 132301 +/- 13386 178551 +/- 16373 30 2007
+/- 470 132238 +/- 1915 182485 +/- 4991 272229 +/- 9325 330434 +/-
47185
Example 9
Real Time PCR Shows IL-21 Expression in Human CD4+ Cells
A. Purified Human T cells as a Primary Source used to Assess Human
IL-21 Expression
[0197] Whole blood (150 ml) was collected from a healthy human
donor and mixed 1:1 with PBS in 50 ml conical tubes. Thirty ml of
diluted blood was then underlayed with 15 ml of Ficoll Paque Plus
(Amersham Pharmacia Biotech, Uppsala, Sweden). These gradients were
centrifuged 30 min at 500 g and allowed to stop without braking.
The RBC-depleted cells at the interface (PBMC) were collected and
washed 3 times with PBS. The isolated human PBMC yield was
200.times.10.sup.6 prior to selection described below.
[0198] The PBMCs were suspended in 1.5 ml MACS buffer (PBS, 0.5%
EDTA, 2 mM EDTA) and 3.times.10.sup.6 cells were set aside for
control RNA and for flow cytometric analysis. We next added 0.25 ml
anti-human CD8 microbeads (Miltenyi Biotec) and the mixture was
incubated for 15 min at 4.degree. C. These cells labeled with CD8
beads were washed with 30 ml MACS buffer, and then resuspended in 2
ml MACS buffer.
[0199] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS magnetic field (Miltenyi). The column was equilibrated
with 5 ml MACS buffer. The isolated primary mouse cells were then
applied to the column. The CD8 negative cells were allowed to pass
through. The column was rinsed with 9 ml (3.times.3 ml) MACS
buffer. The column was then removed from the magnet and placed over
a 15 ml falcon tube. CD8+ cells were eluted by adding 5 ml MACS
buffer to the column and bound cells flushed out using the plunger
provided by the manufacturer. The yield of CD8+ selected human
peripheral T cells was about 51.times.10.sup.6 total cells. The
CD8-negative flow through cells were collected, counted, stained
with anti-human CD4 coated beads, then incubated and passed over a
new VS+ column at the same concentrations as described above. The
yield of CD4+ selected human peripheral T cells was
42.times.10.sup.6 total cells.
[0200] A sample of each of the CD8+ and CD4+ selected human T cells
was removed for staining and sorting on a fluorescence activated
cell sorter (FACS) to assess their purity. A PE-conjugated
anti-human CD4 antibody, an anti-human CD8-FITC Ab, and an
anti-human CD19-CyChrome Ab (all from PharMingen) were used for
staining the CD8+ and CD4+ selected cells. The CD8-selected cells
in this first experiment were 80% CD8+, and the CD4-selected cells
were 85% CD4+. In 2 subsequent experiments (Example 9B), the CD8+
purified cells were 84% and 81% pure, and the CD4+ cells were 85%
and 97% pure, respectively. In one experiment, we stained the
non-binding (flow-through) cells with anti-human CD19-coated beads
(Miltenyi) and ran them over a third magnetic bead column to
isolate CD19+ B cells (these were 92% pure).
[0201] The human CD8+, CD4+ and CD19+ selected cells were activated
by incubating 0.5.times.10.sup.6 cells/ml in RPMI+5% human
ultraserum (Gemini Bioproducts, Calabasas, Calif.)+PMA 10 ng/ml and
Ionomycin 0.5 .mu.g/ml (Calbiochem) for about 4, 16, or 24 hours at
37.degree. C. The T-cells (2.5.times.10.sup.6/well) were
alternately stimulated in 24-well plates pre-coated overnight with
0.5 .mu.g/ml plate-bound anti-CD3 mAb UCHT1 (PharMingen) with or
without soluble anti-CD28 mAb (PharMingen) at 5 .mu.g/ml. At each
timepoint, the cells were harvested, pelleted, washed once with
PBS, and pelleted again. The supernatant was removed and the
pellets were snap-frozen in a dry ice/ethanol bath, then stored at
-80.degree. C. for RNA preparation at a later date.
[0202] Real Time-PCR was performed on these human CD8+, CD4+ and
CD19+ selected cells as described in Example 9B and Example 9C
below for assessing human IL-21 and receptor expression.
B. Primers and Probes for Quantitative RT-PCR for human IL-21
Expression
[0203] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (see, Heid, C A et al.,
Genome Research 6:986-994, 1996; Gibson, U E M et al., Genome
Research 6: 995-1001, 1996; and Sundaresan, S et al., Endocrinology
139:4756-4764, 1998). This method incorporates use of a gene
specific probe containing both reporter and quencher dyes. When the
probe is intact the reporter dye emission is negated due to the
proximity of the quencher dye. During PCR extension using
additional gene-specific forward and reverse primers, the probe is
cleaved by 5' nuclease activity of Taq polymerase which releases
the reporter dye resulting in an increase in fluorescent
emission.
[0204] The primers and probes used for real-time quantitative
RT-PCR analyses were designed using the primer design software
Primer ExpressTM (PE Applied Biosystems). Primers for human IL-21
were designed spanning an intron-exon junction to eliminate
amplification of genomic DNA. The forward primer, ZC22,281 (SEQ ID
NO: 12) and the reverse primer, ZC22,279 (SEQ ID NO12) were both
used at 300 nM concentration to synthesize an 80 bp product. The
corresponding IL-21 TaqMan probe, ZG32 (SEQ ID NO:14) was
synthesized by PE Applied Biosystems. The probe was labeled with a
reporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE Applied
Biosystems) at the 5' end and a quencher fluorescent dye
(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems)
at the 3' end. In order to test the integrity or quality of all the
RNA samples, they were screened for rRNA using the primer and probe
set ordered from PE Applied Biosystems (cat No. 4304483). The
reporter fluorescent dye for this probe is VIC (PE Applied
Biosystems). The rRNA results will allow for the normalization of
the IL-21 results.
[0205] RNA was prepared from pellets provided in Example 9A, using
RNeasy Miniprep.TM. Kit (Qiagen, Valencia, Calif.) per the
manufacturer's instructions. Control RNA was prepared from about 10
million BHK cells expressing human IL-21.
C. Primers and Probes for Quantitative RT-PCR for human zalpha 11
Receptor Expression
[0206] Real time PCR was performed to assess the expression of
IL-21 receptor as per Example 9B and Example 9D, using the cells
prepared under the conditions detailed in 43A, and probes specific
for the IL-21 receptor. The forward primer, ZC22,277 (SEQ ID NO:15)
and the reverse primer, ZC22,276 (SEQ ID NO:16) were used in a PCR
reaction (above) at about 300 nM concentration to synthesize a 143
bp product. The corresponding IL-21 TaqMan.RTM. probe, designated
ZG31 (SEQ ID NO:17) was synthesized and labeled by PE Applied
Biosystems. RNA from BaF3 cells expressing human IL-21 receptor was
used to generate appropriate control for standard curves for the
real-time PCR described in Example 9D below.
D. Real-time Quantitative RT-PCR
[0207] Relative levels of IL-21 RNA were determined by analysis of
total RNA samples using the One-Step RT-PCR method (PE Applied
Biosystems). RNA from BHK cells expressing human IL-21 was used to
generate a standard curve. The curve consisted of serial dilutions
ranging from 2.5-2.5.times.10.sup.-4 ng for the rRNA screen and
25-0.0025 ng for the IL-21 screen with each point analyzed in
triplicate. The total RNA samples were also analyzed in triplicate
for human IL-21 transcript levels and for levels of rRNA as an
endogenous control. Each One-step RT-PCR reaction consisted of 25
ng of total RNA in buffer A (50 mM KCL, 10 mM Tris-HCL, and the
internal standard dye, ROX (PE Applied Biosystems)), appropriate
primers (50 nM for rRNA samples, 300 nM for IL-21 samples) and
probe (50 nM for rRNA, 100 nM for IL-21), 5.5 mM MgCl.sub.2, 300
.mu.M each d-CTP, d-ATP, and d-GTP and 600 .mu.M of d-UTP, reverse
transcriptase (0.25 U/.mu.l), AmpliTaq DNA polymerase (0.025
U/.mu.l) and RNase Inhibitor (0.4 U/.mu.l) in a total volume of 25
.mu.l. Thermal cycling conditions consisted of an initial RT step
at 48.degree. C. for 30 minutes, an AmpliTaq Gold activation step
of 95.degree. C. for 10 minutes, followed by 40 cycles of
amplification for 15 seconds at 95.degree. C. and 1 minute at
60.degree. C. Relative IL-21 RNA levels were determined by the
Standard Curve Method as described in User Bulletin No. 2 (PE
Biosystems; User Bulletin #2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997)
using the rRNA measurements to normalize the IL-21 levels. Samples
were compared relative to the calibrator within each experiment.
The calibrator was arbitrarily chosen based on good quality RNA and
an expression level to which other samples could significantly be
compared. Results of the experiments analyzing the expression of
the IL-21 and IL-21 receptor in stimulated and unstimulated cells
(Example 9A) are as described in Example 9E below.
E. Expression of Human Zalphal11 Receptor and Ligand in CD4+, CD8+
and CD19+ Cells
[0208] The first experiment used RT-PCR, described above, to assess
zalphal11 receptor expression in unstimulated and anti-CD3
stimulated CD4+ and CD8+ samples at timepoints of 0 h (unstimulated
("resting") cells), and at 4 h, 15.5 h and 24 h, after stimulation.
The resting CD4+ sample was arbitrarily chosen as the calibrator
and given a value of 1.00. There was approximately a 4-fold
increase in receptor expression in unstimulated CD4+ cells from 4 h
to 24 h of culture and about an 8-fold increase over the same time
period in anti-CD3 stimulated CD4+ cells. The CD8+ cells showed a
7-fold increase in IL-21 receptor expression that peaked at 4 hrs
and decreased over time. With anti-CD3 stimulation, the CD8+ cells
had a constant 8-fold increase in receptor expression.
[0209] This first experiment also used RT-PCR to assess IL-21
expression in the same anti-CD3 stimulated and unstimulated CD4+
and CD8+ samples. The 4 hr anti-CD3 stimulated CD8+ sample was
arbitrarily chosen as the calibrator and given a value of 1.00. The
results showed that unstimulated CD4+ and CD8+ cells do not express
IL-21 . We observed a significant elevation of expression in the
anti-CD3 stimulated CD4+ cells at 4 h, with about a 300-fold
increase in signal observed at 15.5 h. The CD8+ cells expressed a
small amount of ligand upon anti-CD3 stimulation, however this is
probably due to contamination of the CD8+ population with a small
number of CD4+ cells.
[0210] The second experiment used RT-PCR to assess IL-21 receptor
expression in anti-CD3-stimulated, PMA + Ionomycin-stimulated and
unstimulated CD4+ and CD8+ samples at timepoints of 0 h, and at 3.5
h, 16 h and 24 h after activation. The resting CD8+ sample was
arbitrarily chosen as the calibrator and given a value of 1.00. The
resting CD4+ and CD8+ cells did not have significant amounts of
receptor expression. The expression was about 3 fold higher in the
PMA + Ionomycin-stimulated CD4+ samples at 3.5 h, 16 h and 24 h
after stimulation. The expression in anti-CD3 activated CD4+ cells
peaked at 10-fold above background levels at 3.5 h after
stimulation, then fell back to levels 4-fold above background at 16
h after stimulation. The CD8+ cells showed a 4-fold expression
increase at 3.5 h after PMA + Ionomycin stimulation, with
expression decreasing at subsequent timepoints. As in the first
experiment, the anti-CD3 stimulated CD8+ cells again exhibited an
8-fold above background induction of receptor expression.
[0211] These samples from the second experiment were also used to
assess IL-21 expression. The 24 hr PMA + Ionomycin stimulated CD4+
sample was arbitrarily chosen as the calibrator and given a value
of 1.00. The results showed that again none of the unstimulated
cells expressed IL-21. There was about a 30-fold induction of
ligand expression in the CD4+ cells stimulated with anti-CD3 at 3.5
h, as seen in the previous experiment (at 4 h). However, there was
only about a 5-fold induction with PMA + Ionomycin stimulation at
3.5 h that went down at subsequent timepoints. Again, the CD8+
cells expressed a very small amount of IL-21 that was probably
attributed to contaminating CD4+ cells.
[0212] The final experiment used RT-PCR to assess IL-21 receptor
expression in anti-CD3- and anti-CD3/anti-CD28-stimulated and
unstimulated CD4+ and CD8+ samples at timepoints of 0 h, and at 2
h, 4 h, and 16 h after stimulation. CD 19+ cells activated with PMA
+ Ionomycin were also screened for receptor expression at the same
time intervals. The resting CD4+ sample was arbitrarily chosen as
the calibrator and given a value of 1.00. The 2 h anti-CD3
stimulated CD4+ cells only had a 4-fold induction of receptor,
compared to the 10-fold induction seen at 3.5 h in the previous
experiment. The combination of anti-CD3 and anti-CD28 increased
IL-21 receptor expression to 8-fold above background. The 16 h
anti-CD3/anti-CD28 stimulated CD8+ cells had very low IL-21
receptor expression levels, as seen in the CD8+ cells in previous
experiments (above). The CD19+ cells stimulated with PMA +
Ionomycin had the most significant IL-21 receptor expression with a
19-fold increase at 2 h, but the expression levels decreased back
to those of resting cells by 16 h.
[0213] These samples from the final experiment were also used to
assess IL-21 by RT-PCR. The 16 h anti-CD3/anti-CD28 stimulated CD8+
sample was arbitrarily chosen as the calibrator and given a value
of 1.00. The results showed that at 2 h the CD4+ cells had about a
2-fold induction of IL-21 expression with anti-CD3 stimulation and
a 5-fold induction with anti-CD3 plus anti-CD28 stimulation. These
stimulation conditions induced Ligand expression over time, with
the 16 h stimulated CD4+ cells exhibiting Ligand expression levels
70-fold above background. CD8+ and CD 19+ cells showed no IL-21
expression.
[0214] A certain amount of variation was expected between blood
draws (i.e. multiple samples at different times from the same
patient and between multiple patients). Therefore, data trends were
analyzed within each study or from a single blood sample and the
three experiments above were compared for an overall conclusion.
The trend from the Real Time PCR experiments described above is
that of all the cell types tested, CD19+ B cells activated with PMA
+ ionomycin expressed the highest levels of IL-21 receptor RNA.
CD4+ and CD8+ cells can also be stimulated to express receptor, but
at lower levels than in B cells. IL-21 was expressed almost
exclusively in stimulated CD4+ T cells (and not by CD8+ T cells or
CD19+ B cells). Although stimulation with PMA + Ionomycin induced a
good IL-21 signal in this assay, a significantly higher signal was
obtained from CD4+ T cells stimulated with anti-CD3 mAb or a
combination of anti-CD3 and anti-CD28 mAbs, conditions that better
mimic an antigen encounter in vivo.
Example 10
IL-21-Dependent Proliferation of B-cell Cells Stimulated Anti-CD40
or Anti-IgM
A. Purification of Human B cells
[0215] A vial containing 1.times.10.sup.8 frozen, apheresed human
peripheral blood mononuclear cells (PBMCS) was quickly thawed in a
37.degree. C. water bath and resuspended in 25 ml B cell medium
(RPMI Medium 1640 (JRH Biosciences. Lenexa, Kans.), 10% Heat
inactivated fetal bovine serum, 5% L-glutamine, 5% Pen/Strep)
(Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, Wash.). Cells
were tested for viability using Trypan Blue (Gibco BRL). Ten
milliliters of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology
Inc., Piscataway, N.J.) was layered under the cell suspension and
spun for 30 minutes at 1800 rpm and allowed to stop with the brake
off. The interface was then removed and transferred to a fresh 50
ml Falcon tube, brought up to a final volume of 40 ml with PBS and
spun for 10 minutes at 1200 rpm with the brake on. The viability of
the isolated cells was again tested using Trypan Blue. Alternately
fresh drawn human blood was diluted 1:1 with PBS (Gibco BRL) and
layered over Ficoll/Hypaque Plus (Pharmacia), spun and washed as
above. Cells isolated from either fresh or frozen sources gave
equivalent results.
[0216] B cells were purified from the Ficoll floated peripheral
blood cells of normal human donors (above) with anti-CD19 magnetic
beads (Miltenyi Biotec, Auburn, Calif.) following the
manufacturer's instructions. The purity of the resulting
preparations was monitored by flow cytometric analysis with
anti-CD22 FITC Ab (Pharmingen, San Diego, Calif.). B cell
preparations were typically >90% pure.
B. Purification of Murine B cells
[0217] A suspension of murine splenocytes was prepared by teasing
adult C57B1/6 mouse (Charles River Laboratories, Wilmington, Mass.)
spleens apart with bent needles in B cell medium. RBCs were removed
by hypotonic lysis. CD43 positive cells were removed with CD43
magnetic beads (Miltenyi Biotec) following the manufacturer's
instructions. The purity of the resulting preparations was
monitored by flow cytometric analysis with anti-CD45R FITC Ab
(Pharmingen). B cell preparations were typically >90% pure.
C. Proliferation of Anti-CD40-Stimulated B-Cells in the Presence of
Human or Murine IL-2 1
[0218] The B cells from either the human or mouse source were
resuspended at a final concentration of 1.times.10.sup.6 cells/ml
in B cell medium and plated at 100 .mu.l/well in a 96 well U bottom
plate (Falcon, VWR) containing various stimulation conditions to
bring the final volume to 200 .mu.l/well. For anti-CD40 stimulation
human cultures were supplemented with 1 .mu.g/ml anti-human CD40
(Genzyme, Cambridge, Mass.) and mouse cultures were supplemented
with 1 .mu.g/ml anti-murine CD40 (Serotec, UK). Human or murine
IL-21 was added at dilutions ranging from 1 .mu.g/ml-100 ng/ml. The
specificity of the effect of IL-21 was confirmed by inhibition of
IL-21 with 25 mg/ml soluble human zalpha11 ICEE (Example 10A). All
treatments were performed in triplicate. The cells were then
incubated at 37.degree. C. in a humidified incubator for 120 hours
(human) or 72 hours (mouse). Sixteen hours prior to harvesting, 1
.mu.Ci .sup.3H-thymidine (Amersham, Piscataway, N.J.) was added to
all wells to assess whether the B-cells had proliferated. The cells
were harvested into a 96 well filter plate (UniFilter GF/C,
Packard, Meriden, Conn.) using a cell harvester (Packard) and
collected according to manufacturer's instructions. The plates were
dried at 55.degree. C. for 20-30 minutes and the bottom of the
wells were sealed with an opaque plate sealer. To each well was
added 0.25 ml of scintillation fluid (Microscint-O, Packard) and
the plate was read using a TopCount Microplate Scintillation
Counter (Packard).
[0219] Incubation with IL-21 at concentrations of 3 ng/ml or more
enhanced the proliferation induced by soluble anti-CD40 in a dose
dependent manner in both murine and human B cells by as much as 30
fold. The murine and human B cells responded equally as well to
their respective IL-21. In both species, the stimulation was
specific to IL-21, as it was reversed by the presence of soluble
IL-21 receptor in the culture.
D. Proliferation of Anti-IgM-Stimulated B-Cells in the Presence of
Human or Murine IL-21
[0220] The B cells from either human or mouse source as described
above (Example 10A and Example 10B) were plated as described above
(Example 10C). For anti-IgM stimulation of human cells the plates
were pre-coated overnight with 10 mg/ml F(ab').sub.2 anti-human IgM
Abs (Southern Biotech Associates, Birmingham, Ala.) and washed with
sterile media just prior to use. The cultures were supplemented
with 0-10 ng/ml hu rIL-4 (R&D Systems, Minneapolis, Minn.). For
anti-IgM stimulation of murine cells soluble anti-IgM (Biosource,
Camarillo, Calif.) was added to the cultures at 10 mg/ml. To each
of the preceding anti-IgM/IL-4 conditions, human or murine IL-21
was added at dilutions ranging from 1 pg/ml-100 ng/ml as described
above. The specificity of the effect of IL-21 was confirmed by
inhibition with soluble human zalpha11 receptor as described above
(Example 10C). All treatments were performed in triplicate. The
cells were incubated, labeled with .sup.3H-thymidine, harvested,
and analyzed as described in Example 10C.
[0221] Incubation with IL-21 at concentrations of 0.3 ng/ml or more
inhibited the proliferation induced by insoluble anti-IgM (mouse)
or anti-IgM and IL-4 (human) in a dose-dependent manner. This
inhibition was specific to IL-21 as it was reversed by the presence
of soluble IL-21 receptor in the culture.
Example 11
Human IL-21 Effect on B-Cells and IL-21 Toxic Saporin Fusion
[0222] The effects of human IL-21 were tested on the following
human B-cell lines: and human Burkitt's lymphoma cell lines Raji
(ATCC No. CCL-86), and Ramos (ATCC No. CRL-1596); human EBV B-cell
lymphoma cell line RPMI 1788 (ATCC No. CRL-156); human
myeloma/plasmacytoma cell line IM-9 (ATCC No. CRL159); and human
EBV transformed B-cell line DAKIKI (ATCC No. TIB-206), and HS
Sultan cells (ATCC No. CRL-1484). Following about 2-5 days
treatment with IL-21, changes in surface marker expression were
found in IM-9, Raji, Ramos, and RPMI1788 cell lines, showing that
these cells can respond to IL-21. Human B-cell lines treated with
IL-21 grew much more slowly than untreated cells when re-plated in
cell culture dishes. These cells also had an increased expression
of FAS ligand, as assessed by flow cytometry (Example 11D and
Example 11E), and moderately increased sensitivity to an activating
FAS antibody (Example 11A). This results indicate that IL-21 could
control some types of B-cell neoplasms by inducing them to
differentiate to a less proliferative and or more FAS ligand
sensitive state. Moreover, zalpha11 receptor is expressed on the
surface of several of these cell lines (U.S. Pat. No. 6,307,024).
Thus, IL-21 and the human IL-21-saporin immunotoxin conjugate
(Example 11B, below), or other IL-21-toxin fusion could be
therapeutically used in B-cell leukemias and lymphomas.
A. The Effect of Human IL-21 on B-cell lines.
[0223] IM-9 cells were seeded at about 50,000 cells per ml +/-50
.mu.g/ml purified human IL-21 (U.S. Pat. No. 6,307,024). After 3
days growth the cells were harvested, washed and counted then
re-plated at about 2500 cells/ml in 96 well plates in to wells with
0, 0.033, 0.1 or 0.33 .mu.g/ml anti-FAS antibody (R&D Systems,
Minneapolis). After 2 days an Alamar blue fluorescence assay was
performed (U.S. Pat. No. 6,307,024) to assess proliferation of the
cells.
[0224] IL-21-treated IM-9 cells grew to only 27% the density of the
untreated cells in the absence of anti-FAS antibody. In the
presence of 0.33 .mu.g/ml anti-FAS antibody, the IL-21-treated
cells were inhibited an additional 52% while the untreated cells
were inhibited by only 30%. The overall inhibition of cell growth
with both IL-21 and 0.33 .mu.g/ml anti-FAS antibody treatment was
86%.
[0225] When the IM-9 cells were pretreated for three days with or
without IL-21 and then re-plated at 100 cells per well and grown
with or without anti-FAS antibody for 6 days, the growth of
untreated cells assessed by Alamar Blue assay (U.S. Pat. No.
6,307,024) was inhibited only 25% by anti-FAS antibody while the
growth of IL-21-treated cells was inhibited 95% relative to the
growth of untreated cells in zero anti-FAS antibody.
B. The Effect of Human IL-21 -Saporin Immunotoxin on B-cell
Lines.
[0226] The human IL-21-saporin immunotoxin conjugate
(zalpha11IL-sap) construction and purification is described in
Example 12. The human zalpha11L-sap was far more potent than the
saporin alone in inhibiting cell growth. When the treated cell are
re-plated after a three or four day treatment the human
zalpha11L-sap treated cells grew very poorly.
[0227] IM-9, Ramos and K562 (ATCC No. CCL-243) cells were seeded at
about 2500 cells/well in 96 well plates with zero to 250 ng/ml
human zalpha11L-sap conjugate or 0-250 ng/ml saporin (Stirpe et
al., Biotechnology 10,:405-412, 1992) only as a control. The plates
were incubated 4 days then an Alamar Blue proliferation assay was
performed (U.S. Pat. No. 6,307,024). At the maximal concentration
of human zalpha11-sap conjugate, the growth of IM-9 cells and RAMOS
cells was inhibited by 79% and 65% respectively. K562 cells which
are low/negative by flow for expression of the IL-21 receptor were
not affected by the zalpha11-sap, thus showing the specificity of
the conjugate's effect.
[0228] IM-9 cells were seeded a 50,000 cells/ml into 6 well plates
at zero and 50 ng/ml human zalpha11L-sap conjugate. After 3 days
the cells were harvested and counted then re-plated from 100 to 0.8
cells per well in 2 fold serial dilutions, and 12 wells per cell
dilution without the human IL-21-saporin immunotoxin. After 6 days
the number of wells with growth at each cell dilution was scored
according to the results of an Alamar blue proliferation assay
(U.S. Pat. No. 6,307,024).
[0229] When cell number was assessed, by Alamar blue assay (U.S.
Pat. No. 6,307,024), after 6 days of growth control cells seeded at
about 12.5 and 6.25 cells per well had equivalent growth to
zalpha11-sap treated cells seeded at 100 and 50 cells/well
respectively. Thus, the growth of the surviving treated IM-9 cells
was markedly impaired even after the removal, by re-plating, of the
zalpha11-sap immunotoxin.
[0230] The limited tissue distribution of the human IL-21 receptor
(U.S. Pat. No. 6,307,024 and WIPO Publication Nos. WO 0/17235 and
WO 01/7717), and the specificity of action of the zalpha11-sap to
receptor-expressing cell lines suggest that this conjugate may be
tolerated in vivo.
C. The Effect of Human IL-21 -Saporin Immunotoxin on B-Cell Line
Viability.
[0231] HS Sultan cells (ATCC No. CRL-1484 ) were seeded at about
40,000 cells per ml into 12 well plates and grown for five days
with either no added cytokines or 40 ng/ml purified human IL-21
(U.S. Pat. No. 6,307,024) or 25 ng/ml human zalpha11L-sap conjugate
(Example 12, below) or with 20 ng/ml IFN-alpha (RDI) or IL-21 and
IFN-alpha. IL-21 inhibited the outgrowth of Hs Sultan cells by 63%.
IFN-alpha inhibited the growth by 38%. IL-21 plus IFN-alpha
inhibited growth 78%, indicating that the growth inhibitory effects
of human IL-21 and IFN-alpha may be additive. The human
zalpha11L-sap inhibited growth of the HS Sultans by 92%.
[0232] The results above support the possible use of IL-21 or human
zalpha11L-sap in the treatment of malignancies or other diseases
that express the zalpha11 receptor, particularly those of B-cell
origin. The combination of IL-21 with IFN-alpha is specifically
suggested by their additive effect in the inhibition of HS Sultan
cells. Some other types of lymphoid malignancies and diseases may
also express the IL-21 receptor, as activated T-cells also express
the receptor mRNA (U.S. Pat. No. 6,307,024 and WIPO Publication
Nos. WO 0/17235 and WO 01/7717) and some of these diseases may also
be responsive to IL-21 of IL-21 -toxic fusion therapy.
D. FAS (CD95) Expression on Human B-cell Lines is Increased by
Human IL-21 Stimulation
[0233] Human B-cell lines HS Sultan (ATCC No. CRL-1484), IM-9 (ATCC
No. CRL159), RPMI 8226 (ATCC No. CCL-155), RAMOS (ATCC No.
CRL-1596), DAKIKI (ATCC No. TIB-206), and RPMI 1788 (ATCC No.
CRL-156), were all treated with or without purified 10 to 50 ng/ml
human IL-21 (U.S. Pat. No. 6,307,024) for 2 to 8 days. The cells
were then stained with anti-CD95 PE-conjugated antibody
(PharMingen, San Diego, Calif.), per manufacturer's protocol, and
analyzed on a FACScalibur (Becton Dickinson, San Jose, Calif.). In
all cell lines, anti-CD95 (FAS or APO-1) staining was increased, in
some cases more than two fold, upon treatment with human IL-2
1.
E. FAS (CD95) Expression on Primary Mouse Spleen B-cells is
Increased by Human IL-21 Stimulation
[0234] Primary mouse splenocytes were obtained by chopping up
spleens from 8 to 12 week old C57/BL6 mice. Erythrocytes were lysed
by treating the preparation for 5 seconds with water then put
through a 70 micron sieve. The remaining splenocytes were washed
and plated in RPMI (JRH Bioscience) plus 10% HIA-FBS (Hyclone,
Logan, Utah). IL-2 (R & D Systems) with or without human IL-21,
as described above. They were then incubated at 37.degree. C., in
5% CO.sub.2 for 5 days. The splenocytes were harvested and stained
with anti-CD95 PE conjugated antibody (PharMingen) and anti-CD19
FITC conjugated antibody (PharMingen) per manufacturer's protocol.
The cells were analyzed by flow cytometry on a FACScalibur (Becton
Dickinson). Upon gating on the CD19+ mouse B-cells, it was found
that anti-CD95 staining was increased on B-cells treated with IL-2
plus human IL-21 compared to those in IL-2 alone. The anti-CD95
staining was 37 relative fluorescent units (RFU) on the B-cells in
IL-2 alone and 55 RFU on the B-cells cultured in IL-2 and human
IL-21 .
Example 12
Construction and Purification of IL-21 Toxic Fusion
[0235] Under a supply contract, 10 mg human IL-21 (U.S. Pat. No.
6,307,024) was sent to Advanced Targeting Systems (ATS, San Diego,
Calif.) for conjugation to the plant toxin saporin (Stirpe et al.,
Biotechnology 10,:405-412, 1992). ZymoGenetics received from ATS
1.3 mg of a protein conjugate comprised of 1.1 molecules saporin
per molecule of human IL-21, formulated at a concentration of 1.14
mg/ml in 20 nM Sodium phosphate, 300 nM sodium cloride, pH 7.2.
Example 13
IL-21 Toxic Fusion in vivo
A. Testing IL-21-Saporin Cnjugate in Mice
[0236] IL-21-saporin conjugate (Example 11) was administered to
C57BL6 mice (female, 12 weeks of age, purchased from Taconic) at
two different dosages: 0.5 and 0.05 mg/kg. Injections were given
i.v. in vehicle consisting of 0.1% BSA (ICN, Costa Mesa, Calif.).
Three injections were given over a period of one week (day 0, 2,
and 7). Blood samples were taken from the mice on day 0
(pre-injection) and on days 2 and 8 (post-injection). Blood was
collected into heparinized tubes (Bectin Dickenson, Franklin Lakes,
N.J.), and cell counts were determined using an automated
hematology analyzer (Abbot Cell-Dyn model No. CD-3500CS, Abbot
Park, Ill.). Animals were euthanized and necropsied on day 8
following blood collection. Spleen, thymus, liver, kidney and bone
marrow were collected for histopathology. Spleen and thymus were
weighed, and and additional blood sample was collected in serum
separator tubes. Serum was sent to Pheonix Central Labs, Everett,
Wash., for testing in a standard chemistry panel. Samples were also
collected for flow cytometric analysis as described herein.
[0237] Circulating blood cell counts and serum chemistry
measurements did not differ significantly between IL-21 conjugate
treated mice and mice treated with an equivalent dose of
unconjugated toxin (saporin). Histological analysis of tissues in
IL-21-saporin treated mice showed no significant changes relative
to mice treated with an equivalent dose of unconjugated toxin.
These results indicated that the saporin conjugate was not toxic in
vivo.
B. Testing IL-21 Toxic Samorin Fusion on B-Cell Derived Tumors in
vivo
[0238] The effects of human IL-21 and the human IL-21 toxic saporin
fusion (Example 12) on human tumor cells are tested in vivo using a
mouse tumor xenograft model described herein. The xenograft models
are initially tested using cell lines selected on the basis of in
vitro experiments, such as those described in Example 11. These
cell lines include, but are not limited to: human Burkitt's
lymphoma cell lines Raji (ATCC No.CCL-86), and Ramos (ATCC No.
CRL-1596); human cell line RPMI 1788 (ATCC No. CRL-156); human
myeloma/plasmacytoma cell line IM-9 (ATCC No. CRL159); human cell
line DAKIKI (ATCC No. TIB-206), and HS Sultan cells (ATCC No.
CRL-1484). Cells derived directly from human tumors can also be
used in this type of model. In this way, screening of patient
samples for sensitivity to treatment with IL-21 or with a IL-21
toxic saporin fusion can be used to select optimal indications for
use of zalpha11 in anti-cancer therapy.
[0239] After selection of the appropriate zenograft in vivo model,
described above, IL-21 -induced activity of natural killer cells
and/or IL-21 effects on B-cell derived tumors are assessed in vivo.
Human IL-21 is tested for its ability to generate cytotoxic
effector cells (e.g. NK cells) with activity against B-cell derived
tumors using mouse tumor xenograft models described herein.
Moreover, direct affects of human IL-21 on tumors can be assessed.
The xenograft models to be carried out are selected as described
above. A protocol using IL-21 stimulated human cells is developed
and tested for efficacy in depleting tumor cells and promoting
survival in mice innoculated with cell lines or primary tumors.
Example 14
Preliminary Evaluation of the Aqueous Stability of Human IL-21
[0240] Preliminary studies were conducted to evaluate the aqueous
stability characteristics of human IL-21 in support of
bioprocessing, formulation, and in vivo administration. The
objectives were to: 1) verify the stability and recovery from Alzet
Minipumps & general storage and handling, 2) determine the
stability-indicating nature of several analytical methods including
cation-exchange HPLC (CX-HPLC), reverse-phase HPLC (RP-HPLC), size
exclusion HPLC (SEC-HPLC), & bioassay (BaF3/zalpha11R
proliferation (e.g., U.S. Pat. Nos. 6,307,024), and 3) determine
the stability-limiting degradation pathways and their kinetic
dependencies.
[0241] Aliquots of purified human IL-21 (U.S. Pat. No. 6,307,024)
were prepared by dilution to 2 mg/mL in PBS (pH 7.4) and stored in
low density polyethylene (LDPE) cryovials (Nalgene, 1.8 mL) at
-80.degree. C. (control), 5.degree. C., 30.degree. C., and
37.degree. C. Samples were assayed intermittently over 29 days by
CX-, RP-, SEC-HPLC, and bioassay. Aliquots were also stored at
-80.degree. C. and subjected to freeze-thaw (f/t) cycling
(-80.degree. C./RT; 5.times. f/t, 10.times. f/t). Recovery of human
IL-21 was determined relative to the -80.degree. C. control (1 f/t)
in all assays.
[0242] The remaining human IL-21 solution from the -80.degree. C.
control samples were refrozen (-80.degree. C.) after analysis. This
aliquot (2 f/t) was used to evaluate the thermal and conformational
stability of human IL-21 as a function of pH using circular
dichroism (CD). The 2 mg/mL solution was diluted to 100 .mu.g/mL in
PBS buffers ranging from pH 3.3-8.8. The far-UV CD spectra was
monitored over the temperature range 5-90.degree. C. in 5.degree.
C. intervals (n=3/pH). The CD spectropolarimeter used was a Jasco
715 (Jasco, Easton, Md.). The thermal unfolding was monitored by
changes in ellipticity at 222 nm as a function of temperature.
Estimates of the T.sub.m were estimated assuming a two-state
unfolding model. The data was fit (sigmoidal) using SlideWrite Plus
for Windows v4.1 (Advanced Graphics Software; Encinitas,
Calif.).
[0243] Recovery and stability from Alzet Minipumps (Model No.
1007D; ALZA Corporation, Mountain View, Calif.) was assessed by
filling pumps with 100 .mu.L of the 2 mg/mL human IL-21 solution,
placing the pumps in 1.8 mL LDPE containing 1 mL of PBS (pH 7.4),
and storing them at 37.degree. C. The release/recovery of human
IL-21 from the minipumps was assessed by CX-, RP-, and SEC-HPLC on
days 2, 4, and 7. The activity was assessed by bioassay on day 7.
The study was designed to evaluate the release from 3 pumps per
sampling time.
[0244] The chromatographic data suggested that the CX- &
SEC-HPLC methods were stability-indicating, whereas the RP-HPLC
method was not. At least 3 additional peaks indicating apparent
degradation products were observed by CX-HPLC. The SEC-HPLC method
resolved an apparent human IL-21 aggregate, eluting prior to human
IL-21. However, no significant additional peaks were observed
eluting after the human IL-21 peak. This suggests that the
degradation products observed by CX-HPLC most probably result from
amino acid modifications such as deamidation, rather than
hydrolysis/proteolysis processes leading to clipped variants. A
small degree of fronting/tailing was observed by RP-HPLC (relative
to control) in samples which had been shown to have undergone
significant degradation by SEC- & CX-HPLC. However, apparent
degradation products were not resolved by RP-HPLC. The degradation
observed by CX-HPLC increased as a function of time-temperature,
and followed apparent first-order kinetics. The % human IL-21
recovered by CX-HPLC after 29 days at 37.degree. C., 30.degree. C.,
and 5.degree. C. was 39%, 63%, and 98%, respectively. Aggregation
also increased in a time-temperature dependent fashion. The %
aggregate found in preparations stored for 29 days at 37.degree.
C., 30.degree. C., and 5.degree. C. was 7.4, 3.4, and below
detectable limits (BDL), respectively. No significant differences
were observed by bioassay in any sample, suggesting the degradation
products have equivalent activity to intact human IL-21. No
degradation was observed by any assay in samples subjected to up to
10 f/t cycles.
[0245] The release of human IL-21 from Alzet Minipumps was
consistent with the theoretical expected volume release. This
suggests that significant surface adsorption would not impair the
delivery of human IL-21 using the Alzet Minipumps with a 2 mg/mL
fill concentration. The degradation consistent with that previously
noted was observed. The % purity determined by CX-HPLC of human
IL-21 released after 2, 4, and 7 days was 96%, 90%, and 79%,
repectively. It should be recognized that degradation also occurs
after human IL-21 is released into or diluted with release medium.
Therefore, the % purity within the minipump may be somewhat
different than that determined to be in the release medium. The
bioactivity of each sample was consistent with the expected amount
of human IL-21 released from the minipumps.
[0246] The human IL-21 far-UV CD spectra, as expected, was
consistent with interleukins, such as IL-3 (J. Biochem.,
23:352-360, 1991), IL-4 (Biochemistry, 30:1259-1264, 1991), and
IL-6 mutants (Biochemistry, 35:11503-11511, 1996). Gross changes in
the far-uv CD spectra as a function of pH were not observed.
Results showed that the pH of maximum thermal/conformational
stability was.apprxeq.pH 7.4. Analysis of the unfolding curves were
based on a two-state unfolding mechanism to allow comparison of the
thermal/conformational stability as a function of pH/composition.
However, one or more intermediates may exist during the unfolding
process since the cooperativity was relatively low, based on the
shallowness of the unfolding curve. Although studies were not
specifically designed to determine whether human IL-21 refolds
following thermal unfolding to 90.degree. C., preliminary data
suggests that at least partial refolding occurs after the
temperature of the sample is cooled back to 20.degree. C.
[0247] These studies allow an analytical paradigm to be identified
to evaluate the purity and verify the stability of human IL-21. For
instance, SEC-HPLC can be used to characterize the extent and rate
of aggregation in aqueous solution. Likewise, CX-HPLC can be used
to characterize the extent and rate of degradation of human IL-21
by mechanisms other than aggregation. The bioassay can be used to
verify activity of human IL-21 and it's aqueous degradation
products. For instance, the human IL-21 variants generated in
aqueous solution & resolved by CX-HPLC may themselves be useful
as therapeutic agents, since they have equivalent bioactivity.
Also, the fact that human IL-21 degrades by several different
processes (aggregation, amino acid modifications) suggests a
preferred or unique formulation which minimizes the rate of each
degradation process may be necessary for long-term stability of a
solution product.
[0248] Identification of the nature of the aqueous degradation
products and determination of their kinetic dependencies (pH,
concentration, excipients) is underway. Human IL-21 stability in
serum/plasma is determined to support the design and interpretation
of in vivo studies.
Example 15
IL-21 Effect on B-cell Derived Tumors in vivo
A. Infusion of IL-21 Using Mini-Osmotic Pumps
[0249] Administration of IL-21 by constant infusion via
mini-osmotic pumps resulted in steady state serum concentrations
proportional to the concentration of the IL-21 contained in the
pump. 0.22 ml of human IL-21 (U.S. Pat. No. 6,307,024) contained in
phosphate buffered saline (pH 6.0) at a concentration of 2 mg/ml or
0.2 mg/ml was loaded under sterile conditions into Alzet
mini-osmotic pumps (model 2004; Alza corporation Palo Alto,
Calif.). Pumps were implanted subcutaneously in mice through a 1 cm
incision in the dorsal skin, and the skin was closed with sterile
wound closures. These pumps are designed to deliver their contents
at a rate of 0.25 .mu.l per hour over a period of 28 days. This
method of administration resulted in significant increase in
survival in mice injected with tumor cells (below).
B. IL-21 Effect on B-cell Derived Tumors in vivo
[0250] The effects of human IL-21 (U.S. Pat. No. 6,307,024) were
tested in vivo using a mouse tumor xenograft model described
herein. The xenograft models tested were human lymphoblastoid cell
line IM-9 (ATCC No. CRL159). C.B-17 SCID mice (female
C.B-17/IcrHsd-scid; Harlan, Indianapolis, Ind.) were divided into 4
groups. On day 0, IM-9 cells (ATCC No. CRL159) were harvested from
culture and injected intravenously, via the tail vein, to all mice
(about 1,000,000 cells per mouse). On day 1, mini-osmotic pumps
containing test article or control article were implanted
subcutaneously in the mice. Mice in groups 1-3 (n=9 per group) were
treated with increasing concentrations of IL-21: group 1 contained
2.0 mg/mL of human IL-21 and delivered 12 .mu.g per day; group 2
contained 0.20 mg/mL of human IL-21 and delivered 1.2 .mu.g per
day; group 3 contained 0.02 mg/mL of human IL-21 and delivered 0.12
.mu.g per day. Mice in group 4 (n=9) were a control and were
treated with vehicle (PBS pH 6.0).
[0251] Mice treated with either 12 .mu.g/day or 1.2 .mu.g/day IL-21
infusion had increased survival compared to vehicle treated mice
(p<0.0001 and p<0.005 for 12 .mu.g/day or 1.2 .mu.g/day vs.
vehicle, respectively, using log rank tests of the survival
function). Mice in the 0.12 .mu.g/day dose group had survival no
different than the mice in the vehicle treated group. These results
showed that IL-21 significantly reduced the effects of the B-cell
tumor cells in vivo, significantly resulting in increased
survival.
Example 16
In vivo Anti-Tumor Effects of IL-21 in B16-F 10 Melanoma and EG.7
Thymoma Models
A. Murine IL-21 Effect on B16-F10 Melanoma Metastasis Growth in
vivo
[0252] Mice (female, C57B16, 9 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. On day 0, B16-F10
melanoma cells (ATCC No. CRL-6475) were harvested from culture and
injected intravenously, via the tail vein, to all mice (about
100,000 cells per mouse). Mice were then treated with the test
article or associated vehicle by intraperitoneal injection of 0.1
ml of the indicated solution. Mice in the first group (n=24) were
treated with vehicle (PBS pH 6.0), which was injected on day 0, 2,
4, 6, and 8. Mice in the second group (n=24) were treated with
murine IL-21 (U.S. Pat. No. 6,307,024), which was injected at a
dose of 75 .mu.g on day 0, 2, 4, 6, and 8. Mice in the third group
(n=12) were treated with murine IL-21, which was injected at a dose
of 75 .mu.g daily from day 0 through day 9. All of the mice were
sacrificed on day 18, and lungs were collected for quantitation of
tumor. Foci of tumor growth greater than 0.5 mm in diameter were
counted on all surfaces of each lung lobe. In both groups of mice
treated with murine IL-21, the average number of tumor foci present
on lungs was significantly reduced, compared to mice treated with
vehicle. Mice treated more frequently (i.e. daily) had fewer tumor
foci than mice treated on alternate days, although this was not a
statistically significant finding between these two groups.
[0253] These results indicated that treatment with murine IL-21
either slowed the growth of the B16 melanoma tumors or enhanced the
ability of the immune system to destroy the tumor cells. The
effects of the treatment on tumor cells were likely mediated
through cells of the immune system (i.e. lymphocytes, NK cells),
which do possess receptors for IL-21, such as, for example, IL-21
receptor and zalpha11IL-2R.gamma. (WIPO Publication No.s WO 0/17235
and WO 01/7717) and are known to be associated with anti-tumor
activity.
B. Murine IL-21 Effect on EG.7 Thymoma Growth in vivo
[0254] Mice (female, C57B16, 9 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. On day 0, EG.7
cells (ATCC No. CRL-2113) were harvested from culture and 1,000,000
cells were injected intraperitoneal in all mice. Mice were then
treated with the test article or associated vehicle by
intraperitoneal injection of 0.1 mL of the indicated solution. Mice
in the first group (n=6) were treated with vehicle (PBS pH 6.0),
which was injected on day 0, 2, 4, and 6. Mice in the second group
(n=6) were treated with murine IL-21 (U.S. Pat. No. 6,307,024),
which was injected at a dose of 10 .mu.g on day 0, 2, 4, and 6.
Mice in the third group (n=6) were treated with murine IL-21, which
was injected at a dose of 75 .mu.g on day 0, 2, 4, and 6. In both
groups of mice treated with murine IL-21, time of survival was
significantly increased, compared to mice treated with vehicle. The
group treated with 75 .mu.g doses of IL-21 had significantly
greater survival than the group treated with 10 .mu.g doses, and
33% (2/6 mice) of this group survived longer than 70 days. An
additional portion of this study tested the effect of the same
dosages carried out through day 12. The results were very similar
to the shorter dosing schedule, with both doses having
significantly increased survival over vehicle treatment, and the
highest dose gave the best response (50% survival past 70
days).
[0255] In some experiments about 4,000,000 OT-I T cells were
injected intraperitoneally in the mice on the day prior to day 0.
The mice were then treated with IL-21 or vehicle as above. The
presence of the OT-I T cells had no effect on the survival time of
the vehicle treated mice. In mice treated with IL-21 the presence
of the OT-I T cells enhanced survival time compared to the mice
treated with IL-21 alone.
[0256] These results indicated that treatment with murine IL-21
either slowed the growth of the EG.7 tumors or enhanced the ability
of the immune system to destroy the tumor cells. The increase in
survival conferred by the OT-I T cells in the presence of IL-21
treatment suggests that IL-21 is activating effector cells of the
immune system.
Example 17
IL-21 Effects on Serum Cytokines and Vascular Leak
A. Analysis of IL-21 on Serum Cytokines
[0257] IL-2 therapy is effective in the treatment of certain
cancers. However, the use of IL-2 as a therapeutic agent has been
limited by its toxic effects, namely vascular leak syndrome (VLS).
IL-2 induced VLS is characterized by infiltration of lymphocytes,
monocytes and neutrophils into the lung causing endothelial damage
in the lung eventually leading to vascular leak (reviewed in
Lentsch A B et al, Cancer Immunol. Immunother., 47:243, 1999). VLS
in mice can be induced with administration of repeated high doses
of IL-2 and measuring vascular leak by Evan's Blue uptake by the
lung. Other parameters that have been shown to be characteristic of
VLS in mice include increased serum levels of TNF.alpha. and
IFN.gamma. (Anderson J A et al, J. Clin. Invest. 97:1952, 1996) as
well as increased numbers of activated T, NK and monocytes in
various organs. Blocking of TNF.alpha. with a soluble TNFR-Fc
molecule inhibited lung infiltration by lymphocytes and therefore
lung injury (Dubinett S M et al, Cell. Immunol. 157:170, 1994). The
aim was to compare the ability of IL-2 and IL-21 to induce VLS in
mice and to measure the different parameters indicative of VLS
(Evan's Blue uptake, serum cytokine analysis, spleen cellular
phenotype).
[0258] Mice (female, C57B16, 11 week old; Charles River Labs,
Kingston, N.Y.) were divided into five groups. All groups contained
10 mice per group. Groups are as follows: Group I or Vehicle group
received Phosphate Buffered Saline (PBS); Group II and III received
IL-2 0.6 or 1.8 million IU/injection respectively; Group IV and V
received mouse IL-21 (U.S. Pat. No. 6,307,024) or 100
.mu.g/injection respectively. The study consisted of 4 days, body
weight was measured daily and animals received 7 intraperitoneal
injection of test substance over the 4-day period. Animals received
two daily injections on day 1-3 and on the fourth day received a
single morning injection. Two hours post final injection animals
received a tail vein injection of 1% Evan's blue (0.2 ml). Two
hours post Evan's blue injection mice where anesthetized with
Isoflurane and blood was drawn was serum cytokine analysis.
Following blood draw animals where transcardial perfused with
heparinized saline (25 U hep/ml saline). Following perfusion spleen
was removed and weighed, liver and lung where removed and placed
into 10 mls of formamide for 24 hr incubation at room temperature.
Following 24 hr incubation vascular leakage was quantitated by
Evan's blue extravasation via measurement of the absorbance of the
supernatant at 650 nm using a spectrophotometer.
[0259] Mice were bled and serum separated using a standard serum
separator tube. 25 .mu.l of sera from each animal was used in a
Becton Dickenson (BD) Cytokine Bead Array (Mouse Th1/Th2 CBA Kit)
assay. The assay was done as per the manufacturer's protocol.
Briefly, 25 .mu.l of serum was incubated with 25 .mu.l bead mix
(IL-2, IL-4, IL-5, TNF.alpha. and IFN.gamma.) and 25 .mu.l
PE-detection reagent for two hours at room temperature in the dark.
A set of cytokine standards at dilutions ranging from 0-5000 pg/ml
was also set up with beads as per the manufacturer's instructions.
The incubated beads were washed once in wash buffer and data
acquired using a BD FACScan as per instructions outlined in the
Kit. The data was analyzed using the BD Cytometric Bead Array
Software (BD Biosciences, San Diego, Calif.).
[0260] Serum cytokine analysis using the CBA cytokine kit (Becton
Dickenson, San Diego, Calif.) showed no increases in levels of
IL-2, IL-4, IL-5, IFN.gamma. or TNF.alpha. in the PBS control
treated groups. There was a dose dependent increase in the levels
of IL-5, IFN.gamma. and TNF.alpha. in sera from IL-2 treated mice.
There was no increase in the levels of the 5 measured cytokines in
the serum of mice treated with IL-21. The cytokine levels in the
highest dose of IL-21 mirrored that of the PBS treated animals.
This shows that unlike IL-2 treatment that leads to increase in
serum levels of the inflammatory cytokines IL-5, TNF.alpha. and
INF.gamma., treatment with IL-21 does not have any effect on these
inflammatory cytokines.
[0261] The results from a representative experiment is given in
Table 6. All concentrations are expressed in pg/ml were an average
of 4 animals/group. TABLE-US-00006 TABLE 6 TNF.alpha. IFN.gamma.
IL5 IL4 IL2 PBS 2.4 0.0 2.9 2.2 1.3 IL2 0.6 millU 22.9 21.6 1095.0
2.5 2.0 IL2 1.8 millU 69.1 185.1 1132.9 2.0 1.9 IL2 3.6 millU 78.9
195.6 651.3 1.8 2.1 IL-21 3 .mu.g 2.1 1.6 2.7 1.7 1.6 IL-21 100
.mu.g 3.1 0.0 4.0 0.0 1.1 IL-21 200 .mu.g 7.3 1.9 4.0 2.6 1.9
[0262] As shown in Table 5 above, treatment of mice with IL-2
resulted in a dramatic increase in serum inflammatory cytokines,
namely IL-5, IFN.gamma. and TNF.alpha.. Treatment of mice with
IL-21 did not show any increase in cytokine levels above PBS
treated mice. These results show that even at the highest doses,
IL-21 does not upregulate inflammatory cytokines and it's effect on
cells in vivo is different from IL-2.
[0263] Treatment of mice with repeated high dose IL-2 resulted in
increased serum levels of IL-5, IFN.gamma. and TNF.alpha.. These
pro-inflammatory cytokines have been shown to play a role in VLS
associated with IL-2 toxicity. Blocking TNFa resulted in decreased
lymphocyte infiltration into the lungs and decreased lung injury
associated with IL-2 toxicity (Dubinett S M et al, 1994, Cell.
Immunol. 157:170). IL-21 treatment did not have any effects on
serum IL-5, TNF.alpha. or IFN.gamma. levels. This suggests that
IL-21 acts different from IL-2 in vivo and that the lack of
pro-inflammatory cytokines in sera of IL-21 treated mice might
indicate lesser toxicity of IL-21 compared to IL-2.
B. Analysis of IL-21 on Vascular Leak--Immunophenotyping of Splenic
Cells
[0264] IL-2 induced vascular leak syndrome (VLS) involves organ
damage that occurs at the level of postcapillary endothelium.
However, this damage occurs secondary to two distinct pathological
processes: the development of VLS, and transendothelial migration
of lymphocytes. Acute organ injury is mediated by infiltrating
neutrophils while chronic organ injury is mediated by infiltration
monocytes and lymphocytes (reviewed in Lentsch A B et al, supra.).
In mice, depletion of cells with surface phenotypes characteristic
of LAK or NK cells ameliorates organ damage (Anderson T D et al,
Lab. Invest. 59:598, 1988; Gately, M K et al. J. Immunol., 141:189,
1988). Increased numbers of NK cells and monocytes is therefore a
marker for IL-2 mediated cellular effects of VLS. In addition, IL-2
directly upregulates the expression of adhesion molecules (i.e
LFA-1, VLA-4 and ICAM-1) on lymphocytes and monocytes (Anderson J A
et al, supra.). This increase is thought to enable cells to bind
activated endothelial cells and help in transmigration of cells to
the tissue. Increased expression of these molecules is considered
another marker of IL-2 induced cellular activation during VLS. The
aim of this study was to study splenic cells from IL-2 and IL-21
treated mice under a VLS protocol and compare the effects of the
two cytokines to mediate cellular effects associated with VLS.
[0265] Groups of age and sex matched C57BL/6 mice treated and
described above (Example 17A) were analysed. On d4, mice were
sacrificed and phenotype of splenic cell populations studied by
standard flow cytometry. Splenic weight and cellularity were
dramatically increased in IL-2 treated mice compared to PBS treated
mice. IL-21 treated mice had a slight increase in splenic weights
(at the higher doses) but no significant increase in splenic
cellularity compared to the PBS treated groups. Cell population
analysis showed a significant increase in the percentage and
numbers of NK, NKT and monocytes in IL-2 treated mice but not in
the IL-21 treated mice. Furthermore, there was a dose dependent
dramatic increase in LFA-1 expressing cells in the IL-2 treated
groups compared to PBS controls. IL-21 treatment had no effect on
LFA-1 expression on splenic cells.
[0266] Spleens were isolated from mice from the various groups. Red
blood cells were lysed by incubating cells for 4 minutes in ACK
lysis buffer (0.15M NH4Cl,1 mM KHCO3, 0.1 mM EDTA) followed by
neutralization in RPMI-10 media (RPMI with 10% FBS). The expression
of cell surface markers was analyzed by standard three color flow
cytometry. All antibodies were obtained from BD Pharmingen (San
Diego, Calif.). Fluorescin-isothiocyanate (FITC) conjugated CD11a
(LFA-1), CD49d (VLA-4, a chain), Gr-I FITC, phycoerythrin (PE)
conjugated CD4, NK1.1, CD11b and CyC-conjugated CD8, CD3 and B220
were used to stain cells. 1-3.times.10.sup.6 cells were used for
individual stains. Non-specific binding was blocked by incubating
cells in blocking buffer (PBS, 10% FBS, 20 ug/ml 2.4G2). After
blocking, cells were incubated with primary antibodies for 20
minutes. Unless specified otherwise, all mAbs were used at
lug/stain in a volume of 100 ul. Cells were washed once in
1.times.PBS and resuspended in PBS before being acquired using the
FACScan or FACSCalibur instruments (BD Biosciences, San Diego,
Calif.). Data was analyzed using the Cellquest Software (BD
Biosciences).
[0267] IL-2 treated mice had significantly increased spleen weights
compared to PBS treated groups (Table 7, below) IL-21 treated mice
had significant increase in spleen weight over controls. However,
the increases in IL-21 treated groups were significantly less than
in the IL-2 treated groups (p=0.0002). The increase in spleen
weights in both groups was dose dependent. Table 7 below shows the
treatment groups; the average splenic weights were shown in mg, and
n=4. TABLE-US-00007 TABLE 7 Total spleen weight (mg) Stdev p value
(vs PBS) PBS 63.5 9.7 0 IL-2 (0.6) 177.5 17.8 <0.0001 IL-2 (1.8)
204.25 10 <0.0001 IL-2 (3.6) 231.2 9.6 <0.0001 IL-21 (33)
92.8 6 0.0022 IL-21 (100) 117.6 19.3 0.0024 IL-21 (200) 125.85 33
0.0111
[0268] Average Splenic cellularity data is shown in Table 8, below
(n=4). Higher dose IL-2 treatment increased splenic cellularity
significantly over control PBS treated groups. IL-21 treated groups
did not show significant increase in splenic cellularity compared
to PBS groups. TABLE-US-00008 TABLE 8 Total cells (.times.10.sup.6)
Stdev p value (vs PBS) PBS 48 16.4 o IL-2 (0.6) 57.1 11.8 0.4014
IL-2 (1.8) 100.4 21.6 <.0083 IL-2 (3.6) 101.8 4.25 <.0007
IL-21 (33) 58.8 13.5 0.3463 IL-21 (100) 48 7.83 0.9769 IL-21 (200)
53.8 22.5 0.6917
[0269] IL-2 induced VLS is characterized by increased numbers of NK
cells, monocytes and cells expressing the adhesion marker LFA-1
(reviewed in Lentsch A B et al, supra.). The above data using IL-2
reproduces published reports on the increase of NK cells, monocytes
and LFA-1+ cells. IL-2 treated mice show all signs of VLS compared
to controls. In contrast, IL-21 treated mice, although having an
increased uptake of Evan's Blue, show no increase in serum
pro-inflammatory cytokines, or increase in LFA-1+ cells or NK
cells. Furthermore, although IL-21 treated mice do show increased
numbers of monocytes, the increase is less than what is seen with
IL-2 treated animals, suggesting that IL-2 mediated effects are
more severe than IL-21 mediated effects. Taking together the
splenic cellularity data and the serum cytokine data, IL-21 does
not induce a comparable inflammatory response as IL-2. All
parameters analyzed would indicate that IL-21 induces minor if any
inflammatory response when administered in a VLS protocol in
similar doses to IL-2 (weight/weight).
[0270] In addition, as shown in Table 9 and Table 10, below, flow
cytometry analysis of spleen cells from mice revealed that IL-2
treated mice had a dose dependent increase in the % and numbers of
splenic NK/T cells (NK1.1+CD3+), NK cells (NK1.1+CD3-), macrophages
(CD11+) and LFA-1+ cells (TABLE III and IV). IL-21 treated mice had
no increase in NK/T cells, NK cells or LFA-1+ cells. There was an
increase in the % and numbers of macrophages and granulocytes (data
not shown) in IL-21 treated group compared to the control PBS
treated groups. This increase was similar or less than the increase
in IL-2 treated mice. TABLE-US-00009 TABLE 9 Average % of lineage
cells in spleen (n = 4) % % LFA- % NK/T % NK macrophages % B % CD4
T % CD8 T 1+ PBS 0.7225 3.1925 6.625 50.025 21.975 14.275 11.1775
IL-2 (0.6) 4.34 9.3375 12.525 43.9 17.65 10.15 29.26 IL-2 (1.8) 3.2
13.9 14.525 43.75 15.55 11.55 34.825 IL-2 (3.6) 3.075 14.3 11.875
42.8 14.875 17.325 44.05 IL-21 (33) 0.615 2.9875 7.825 53.65 17.925
10.95 8.4375 IL-21 (100) 0.63 2.76 11.375 48.325 17.825 11.275
13.35 IL-21 (200) 1.025 3.4325 16.175 45.125 17.225 12.15 15.7
[0271] TABLE-US-00010 TABLE 10 Splenic cell numbers
(.times.10.sup.6 cells, n = 4) NKT NK CD11b B220 Cd4 Cd8 LFA-1 Gr-1
PBS 0.33 1.54 3.19 24.34 10.19 6.58 5.41 1.11 IL-2 (0.6) 2.34 5.33
7.04 25.23 10.16 5.77 16.60 2.82 IL-2 (1.8) 3.08 14.29 14.16 43.63
15.57 11.62 35.11 7.65 IL-2 (3.6) 3.15 14.59 12.08 43.53 15.13
17.64 44.83 4.76 IL-21 (33) 0.37 1.77 4.67 31.50 10.47 6.32 5.05
1.05 IL-21 0.30 1.33 5.35 23.16 8.55 5.40 6.35 1.54 (100) IL-21
0.56 1.86 8.46 23.85 9.20 6.39 8.43 3.20 (200)
[0272] In addition, additional endpoints were measured between
groups. The following endpoints where compared: Body weight, spleen
weight, vascular leakage in lung and liver, and serum cytokines. No
significant difference in body weights was observed between groups.
As discussed above, animals treated with both doses of IL-2, Group
II and III, had significantly heavier spleen weights as compared to
IL-21 and PBS control treated animals (p<0.0001). Animals
treated with both doses of IL-21 treated animals, Group IV and V,
had significantly heavier spleen weights as compared to PBS control
animals (p<0.007 Group IV and p<0.0001 Group V).
[0273] Vascular leakage was also measured in both lung and liver.
In lung, both groups of IL-2 treated animals, Group II and III, had
a significant increase in vascular leakage (p<0.0001) as
compared to PBS control animals. Only Group III, the high dose of
IL-2 had a significant increase in vascular leakage as compared to
both low dose and high dose IL-21 (p<0.0001 and p<0.0065)
respectively. Only the highest dose of IL-21, Group V, had a
significant increase in vascular leakage as compared to PBS treated
animals (p<0.0001). However, the amount of vascular leak was
significantly lower than all of the IL-2 treated animals. In liver,
both the low and high dose of IL-2 treated animals had a
significant increase in vascular leakage (p<0.0016 and
p<0.0001 respectively) as compared to PBS treated animals.
Animals treated with the high dose of IL-2 had a significant
increase in vascular leakage as compared to both low and high dose
IL-21 treated animals (p<0.0002 and p<0.0001 respectively).
Only the low dose IL-21 treated animals had a significant increase
in vascular leakage as compared to PBS treated animals
(p<0.0397).
[0274] Example 18
Flow Cvtometric Analysis IL-21 Receptor Expression.
[0275] The expression of IL-21 receptors on neoplastic B cells
derived from non-Hodgkin's lymphoma (HL) specimens was assessed.
Multiple MAbs were used to identify neoplastic B cells and to
co-localize IL-21 receptors (WIPO Publication No.s WO 0/17235 and
WO 01/77171). The immunofluorescent staining by anti-IL-21R MAb or
by biotin-IL-21 was recorded as mean peak fluorescence. The
qualitative scores were assessed based on the shift in mean peak
fluorescence relative to an isotype matched control MAb.
[0276] Using either anti-IL-21 receptor MAb or biotin-IL-21 (U.S.
Pat. No. 6,307,024) we consistently detected IL-21 receptor on
follicular lymphoma (FL) specimens derived from lymph node.
However, nearly all specimens derived from chronic lymphocytic
leukemia (CLL) patients did not show significant staining for IL-21
receptors, or staining at very low intensity relative to negative
control MAbs. The staining by anti-IL-21 receptor MAbs and
biotin-IL-21 correlated well and detected moderate staining of
follicular lymphoma. These data suggested that IL-21 receptors
represent a therapeutic target for follicular lymphoma.
Example 19
Activity of Mouse IL-2 1-Treated Alloreactive Murine CTL
(cytotoxicity assays)
A. CTL Assay
[0277] CTL (cytotoxic T lymphocyte)-mediated target cytolysis was
examined by a standard .sup.51Cr-release assay. Alloreactive
(H-2.sup.b anti-H-2.sup.d) CTL were generated in a mixed lymphocyte
culture with C57B1/6 splenocytes (H-2.sup.b) with 3000
rad-irradiated Balb/c splenocytes (H-2.sup.d). After 7 days, the
CTL were re-stimulated with irradiated Balb/c splenocytes (and no
additional cytokines). After an additional 7 days, CTL were
re-stimulated for 5 days in the presence of supernatants collected
from conA-activated rat splenocytes (a crude source of cytokines
known to support CTL growth), 10 ng/ml recombinant mouse IL-2
(R&D Systems, Inc, Minneapolis, Minn.), recombinant human IL-15
(R&D Systems), IL-21, or a combination of IL-15 and IL-21 (5
ng/ml each). After 5 days, the CTL were assayed for their capacity
to lyse .sup.51Cr-labeled target cells: H-2.sup.d P815 mastocytoma
cells (ATCC No. TIB-64) and the H-2.sup.b thymoma EL4 (ATCC No.
TIB-39) as a negative control.
[0278] We grew P815 and EL4 cells in RP10 medium (standard RPMI
1640 (Gibco/BRL, Grand Island, N.Y.) supplemented with 10% FBS
(Hyclone) as well as 4 mM glutamine (Gibco/BRL), 100 I.U./ml
penicillin+100 MCG/ml streptomycin (Gibco/BRL), 50 .mu.M
.beta.-mercaptoethanol (Gibco/BRL) and 10 mM HEPES buffer
(Gibco/BRL). On the day of assay, 1-2.times.10.sup.6 target cells
were harvested and resuspended at 2.5-5.times.10.sup.6 cells/ml in
RP10 medium. We added 50-100 .mu.l of 5 mCi/ml .sup.51Cr-sodium
chromate (NEN, Boston, Mass.) directly to the cells and incubated
them for 1 hour at 37.degree. C., then washed them twice with 12 ml
of PBS and resuspended them in 2 ml of RP10 medium. After counting
the cells on a hemacytometer, the target cells were diluted to
0.5-.times.10.sup.5 cells/ml and 100 .mu.l (0.5-.times.10.sup.4
cells) were mixed with effector cells at various effector:target
ratios. After a 4-hour co-incubation of effector cells and the
labeled target cells at 37.degree. C., half of the supernatant from
each well was collected and counted in a gamma counter for 1
min/sample. The percentage of specific .sup.51Cr release was
calculated from the formula 100.times.(X-Y)/(Z-Y), where X is
.sup.51Cr release in the presence of effector cells, Y is the
spontaneous release in the absence of effectors, and Z is the total
.sup.51Cr release from target cells incubated with 0.5% Triton
X-100. Data were plotted as the % specific lysis versus the
effector-to-target ratio in each well.
[0279] CTL re-stimulated in the presence of rmIL-2 exhibited the
highest lytic activity on the P815 target cells, achieving >70%
specific lysis at an effector-to-target ratio of 33:1. The next
most active CTL were those re-stimulated in the presence of
IL-21+rhIL-15 (62% specific lysis), followed by CTL cultured with
rhIL-15 (.about.50% lysis), CTL cultured with IL-21 alone (30%
lysis), and CTL re-stimulated with rat conA supernatant (.about.10%
lysis). None of the CTL lysed the H-2.sup.b EL4 cells (all CTL
lysed fewer than 2% of the EL4 targets, even at the highest
effector-to-target ratio of 33:1). This pattem of cytokine
enhancement of cytolysis
(IL-2>IL-21+IL-15>IL-15>IL-21>conA SN) held true in 2
replicate experiments. These data demonstrate that IL-21,
particularly in combination with IL-15, can enhance CTL effector
function.
Example 20
Delayed Type Hypersensitivity in IL-21 Knockout (KO) Mice
[0280] IL-21 is a cytokine that is produced by T cells and has been
shown to play a role in T cell proliferation and function. Delayed
Type Hypersensitivity (DTH) is a measure of helper CD4 T cell
responses to specific antigen. In this, mice are immunized with a
specific protein (E.g., chicken ovalbumin, OVA) and then later
challenged with the same antigen in the ear. Increase in ear
thickness after the challenge is a measure of specific immune
response to the antigen, mediated mainly by CD4 T cells. To
understand the in vivo function of IL-21, mice deficient in IL-21
protein were engineered (IL-21 KO mice). If IL-21 is important for
T cell responses, IL-21 KO mice should be expected to have a defect
in T cell responses. One method to test this is to induce a DTH
response in IL-21 KO mice. IL-21 KO mice and control liffermates
were immunized with OVA mixed with an adjuvant CFA (Complete
Freund's Adjuvant). Groups of mice were then rechallenged in the
ear with either PBS (control) or OVA. Control mice developed good
DTH when injected with OVA as shown by increase in the ear
thickness at 24 hours post challenge. In contrast, IL-21 KO mice
had a lesser degree of ear thickness compared to controls. This
difference was statistically significant (p=0.0164). However, at 48
hour post challenge, there was no difference in the response of
wild type or IL-21 KO mice. As expected, both control and IL-21 KO
mice did not respond to PBS (no change in ear thickness).
[0281] IL-21 KO mice (n=8) and control wild type littermates (n=8)
were immunized in the back with 100 .mu.g chicken ovalbumin (OVA)
emulsified in CFA in a total volume of 200 .mu.l. Seven days after
the immunization, half the mice in each group (n=4/gp) were
injected with 10 .mu.l PBS in the ear and the other half injected
with 10 .mu.g OVA in PBS in a volume of 10 .mu.l. Ear thickness of
all mice was measured before injecting mice in the ear (0
measurement). Ear thickness was measured 24 hours and 48 hours
after challenge. The difference in ear thickness between the 0
measurement and the 24 hour or 48 hour measurement was
calculated.
[0282] At 24 hour post challenge, control mice or IL-21 KO mice
rechallenged with PBS showed minimal or no change in ear thickness.
In response to the OVA rechallenge, control mice ears showed
significant inflammation (7.3.+-.1.1.times.10.sup.-3 in). In
contrast, IL-21 KO mice showed a decrease in ear thickness compared
to controls (5.+-.0.42.times.10.sup.-3 in). This difference was
statistically significant (p=0.0164). This suggests that IL-21 does
play an important role in CD4 T cell responses. However, at 48 hour
post challenge, responses in IL-21 KO mice were no different from
controls suggesting that IL-21 does not influence the response at
this stage. Further experiments are underway to assess the role of
IL-21 in DTH responses and in T cell responses.
[0283] These results suggest that IL-21 plays an important role in
CD4 T cell responses. CD4 T cell responses contribute significantly
to immunity, both in a positive manner to boost immunity towards
microbes and tumors and in a negative manner in cases of
autoimmunity and inflammation. Use of IL-21 may be considered to
boost CD4 T cell responses based on the above results.
Example 21
IL-21 Modifies the Response of OT-I T cells to OVA Peptide as
Presented by Murine
Dendritic Cells.
A. Isolation and Labeling of OT-I T Cells
[0284] Mice bearing a transgenic T cell receptor specific for
OVA257-264 in H-2K.sup.b are available (OT-I transgenics, Jackson
Laboratories). Cells from lymph nodes from these animals were
adherence depleted and the CD8 T cells (OT-I T cells) were enriched
by negative selection using CD8 Cellect columns (Cedarlane
Laboratories, Homby, Ontario, Canada). Purity of CD8 T cells was
assessed by flow cytometry and was typically 90-95% with <1% CD4
T cells.
[0285] OT-I T cells were labeled with carboxyfluorescein diacetate
succinimidyl ester (CFSE; Molecular Probes, Eugene, Oreg.) by
placing them in growth media comprising RPMI-1640 medium
supplemented with 10% FCS (JRH, Lenexa K S; Hyclone, Logan Utah), 2
mM glutamine (Gibco BRL), 50 U/ml penicillin (Gibco BRL), 50
.mu.g/ml streptomycin (Gibco BRL, Grand Island, N.Y.) and 50 .mu.M
2-mercaptoethanol (Sigma, St Louis, Mo.) containing 5 .mu.M CFSE
for 5 min at room temperature. The cells were then washed three
times, each time by resuspending in PBS containing 5% FBS,
centrifuging 5 min at 300' g, 20.degree. C., and removing the
supernatant. Cells were resuspended in growth media prior to
use.
B. Preparation of Murine Dendritic Cells
[0286] Bone marrow derived dendritic cells (DCs) from mouse bone
marrow were cultured in growth media the presence of GM-CSF using
well-known methods (e.g., Inaba, K. et al., J. Exp. Med.
176:1693-1702, 1992). After six days in culture they were
stimulated with 1 .mu.g/ml LPS (Sigma, St Louis, Mo.) overnight and
then washed in growth media prior to use.
C. In vitro Stimulation of T Cells
[0287] DCs prepared as above were pulsed with 10 nm OVA257-264
peptide (SEQ ID NO:17) for 2 hours. The pulsed DCs are washed in
growth media to remove any unbound peptide and then cultured with
purified OT-I T cells prepared as described above in the presence
of either media alone or 20 ng/ml mouse rIL-2 (R&D Systems,
Minneapolis, Minn.) or 50 ng/ml murine IL-21 (U.S. Pat. No.
6,307,024). After either 48 or 72 hours of incubation the cells
were harvested and analyzed by flow cytometry for levels of CFSE
fluorescence and Annexin V binding (Pharmingen, San Diego, Calif.)
per manufacturers instruction.
[0288] Results showed that when OT-I T cells are presented specific
antigen on DC's they undergo 3-5 rounds of cell division by day 2
and 5-7 rounds of cell division by day 3 as evidenced with CFSE
labeling. In the presence of IL-2 their proliferation is increased
such that by day 2 they have gone 5-6 rounds and by day 3, 7-9
rounds. When the T cells are treated with IL-2 they undergoing
apoptosis at day 3 as demonstrated by Annexin V binding. In
contrast to IL-2, IL-21 induces increased T cell proliferation and
prevents Annexin V labeling up to day 3. IL-21 continues to enhance
proliferation and prevent apoptosis even in the presence of added
IL-2.
[0289] IL-21 both enhances proliferation and reduces apoptosis of
the murine CTL cells. This activity implies a positive
immunostimulatory role for IL-21 in clinical settings, such as
cancer or viral disease, where CTL's can play a role.
Example 22
Murine IL-21 Effect on EG.7 Thymoma Growth in vivo:
IL-21 Modifies the Response of OT-I T Cells in the EG-7 Model of
CTL Mediated Anti-Tumor Activity
[0290] Cytotoxic T lymphocytes (CTL) recognize infected and
transformed cells by virtue of the display of viral and tumor
antigens on the cell surface. Effective anti-tumor responses
require the stimulation and expansion of antigen specific CTL
clones. This process requires the interaction of several cell types
in addition to CTL and usually results in the establishment of
immunologic memory. The EG-7 tumor cell line is transfected with
chicken ovalbumin and thereby expresses a well characterized T cell
antigen, an ova peptide (SEQ ID NO:17) presented in H-2K.sup.b.
OT-I T cells (Example 21) kill EG7 tumor cells in vitro and in
vivo. (Shrikant, P and Mescher, M. J. Immunology 162:2858-2866,
1999).
[0291] Mice (female, C57B16, 9 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. On day 0, EG.7
cells (ATCC No. CRL-2113) were harvested from culture and 1,000,000
cells were injected intraperitoneal in all mice. Mice were then
treated with the test article or associated vehicle by
intraperitoneal injection of 0.1 ml of the indicated solution. Mice
in the first group (n=6) were treated with vehicle (PBS pH 6.0),
which was injected on day 0, 2, 4, and 6. Mice in the second group
(n=6) were treated with murine IL-21, which was injected at a dose
of 10 .mu.g on day 0, 2, 4, and 6. Mice in the third group (n=6)
were treated with murine IL-21, which was injected at a dose of 75
.mu.g on day 0, 2, 4, and 6. In both groups of mice treated with
murine IL-21, time of survival was significantly increased,
compared to mice treated with vehicle. The group treated with 75
.mu.g doses of IL-21 had significantly greater survival than the
group treated with 10 .mu.g doses, and 33% (2/6 mice) of this group
survived longer than 70 days. An additional portion of this study
tested the effect of the same dosages carried out through day 12.
The results were very similar to the shorter dosing schedule, with
both doses having significantly increased survival over vehicle
treatment, and the highest dose gave the best response (50%
survival past 70 days).
[0292] In some experiments 4,000,000 OT-I T cells were injected
intraperitoneal in the mice on the day prior to day 0. The mice
were then treated with IL-21 or vehicle as above. At various times
after treatment OT-I T cells were recovered from the peritoneal
cavity and counted. The presence of the OT-I T cells had no effect
on the survival time of the vehicle treated mice. IL-21 treatment
resulted in a ten-fold increase in the number of OT-I T cells that
could be recovered from the peritoneal cavity. In mice treated with
IL-21 the presence of the OT-I T cells enhanced survival time
compared to the mice treated with IL-21 alone.
[0293] The increase in survival conferred by IL-21 treatment with
or with out exogenously added tumor specific T cells shows that
IL-21 is activating endogenous effector cells of the immune system.
The increased recovery of OT-I T cells from the peritoneal cavity
shows that IL-21 is increasing the number of tumor specific T cells
at the site of the tumor.
[0294] As predicted by the ability of IL-21 to enhance T cell
survival in vitro these results indicate that treatment with IL-21
has enhanced the ability of the immune system to destroy tumor
cells in vivo. These results in vivo demonstrate a positive
immunostimulatory role for IL-21 in relevant clinical settings,
such as in human cancer or viral disease, where CTL's can play a
role in combating disease.
Example 23
IL-21 Reduces Tumor Load in the RMA-RAE1 Model of NK Mediated
Anti-Tumor Activity
[0295] Natural killer cells serve as a first line of defense
against certain viral infections and tumors. Effective NK cell
activity does not require prior exposure to the target nor are they
thought to maintain immunologic memory of the target. Thus, NK
cells "sense" if cells are transformed, infected, or otherwise
"stressed" by virtue of a range of molecules on the surface of the
target cell. RAE-1 is a protein expressed on the surface of
"stressed" cells which specifically engages an activating a
receptor on the surface of NK cells thereby leading to lysis of the
"stressed" cell. Transfection of the RMA tumor cell line with RAE-1
renders it sensitive to lysis by NK cells both in vitro and in
vivo.
[0296] The RMA lymphoma cell line (provided by Dr. L. Lanier and
Dr. Jay Ryan, UCSF, San Francisco, Calif.) was grown in RPMI-1640
medium supplemented with 10% FCS (JRH, Lenexa K S; Hyclone, Logan
Utah), 2 mM glutamine (Gibco BRL), 50 U/ml penicillin (Gibco BRL),
50 .mu.g/ml streptomycin (Gibco BRL, Grand Island, N.Y.) and 50
.mu.M 2-mercaptoethanol (Sigma, St Louis, MO). Stable transfectants
of RMA-RAE-1delta or mock-transfected RMA cells were established by
electroporation: 30 .mu.g of RAE-1delta-pCDEF3 plasmid (RAE-1delta
transfectant), or pCDEF3 plasmid (mock-transfectant) was added to
about 1.times.10.sup.7 cells in RPMI-1640 medium in a 4-mm cuveffe
(BioRad, Richmond, Calif.), respectively. The pCDEF3 vector was
kindly provided by Dr. Art Weiss (UCSF, San Francisco Calif.).
Electroporation was performed by using a BioRad gene pulser (250 V,
960 .mu.F). 48 h after electroporation, RMA-RAE-1delta and
mock-transfected cells were cultured in complete RPMI-1640 medium
supplemented with 1 mg/ml G418 (GIBCO BRL).
[0297] Groups of six or more animals per experiment were injected
intraperitoneally with cells which were mock-transfected or
transfected with RMA-RAE-1delta. Preliminary experiments titrating
the number of tumor cells injected indicated that 1.times.10.sup.4
RMA cells and 1.times.10.sup.5 RMA-RAE-1delta cells resulted in
tumor formation and subsequent morbidity in 100% of animals.
Intraperitoneal (IP) inoculation of mice with RMA-RAE1delta cells
at numbers comparable to lethal doses (about 1.times.10.sup.4) of
the parental RMA cells results in the tumors being completely
rejected without the involvement of T cells or the establishment of
immunologic memory. IP inoculation of mice with a 10 fold excess of
RMA-RAE1delta cells results in death of the mouse presumably by
`swamping` the capacity of the NK cells to reject the tumor
(Cerwenka et al., Proc. Nat. Acad. Sci. 98:11521-11526, 2001). For
cytokine efficacy experiments six IP injections of 10.mu.g of
murine IL-21 or vehicle control were administered to the mice every
other day on days -4, -2, 0, 2, 4 and 6. All mice were monitored
daily for tumor ascites development, indicated by swelling of the
abdomen, and were sacrificed when tumor burden became excessive to
avoid pain and suffering. Animals were regarded tumor free when
surviving longer than 8 weeks. For the re-challenge experiments,
surviving animals were inoculated with 1.times.10.sup.4 RMA-mock
cells after 8 weeks.
[0298] The administration of small amounts of murine IL-21 resulted
in enhanced survival of mice receiving the 10 fold excess number of
RAEI bearing tumor cells. Some IL-21 treated mice became completely
tumor-free. IL-21 treatment had no effect on the survival of mice
given the parental RMA cell line showing that the effect was
specifically mediated by NK cells. It appeared that RAEI, and
thereby NK cells, were required for the IL-21 effect. Moreover,
mice that survived the lethal RMA-RAE1 tumor challenge by virtue of
IL-21 treatment were able to reject a subsequent challenge with the
parental RMA cell line. This showed that IL-21 had also induced
immunological memory in these mice. This ability of IL-21 to
enhance the activity of NK cells shows that IL-21 can have
therapeutic benefit in the treatment of patients who have tumors or
viral diseases.
Example 24
Immunohistochemistry of IL-21 in Various Human Tissues and Cell
Lines
[0299] The purpose of this experiment was to determine if IL-21
could be detected in select tissues by means of
immunohistochemistry. Tissues were processed by standard
immunohistochemical methods using a Techmate 500 (BioTek Solutions,
Tucson, Ariz.). Briefly, deparaffinized sections of
paraffin-embedded tissues were treated with 5% normal goat serum in
PBS and a blocking agent (Zymed Laboratories, Inc., South San
Francisco, Calif., Reagent A and B (ready to use)) to minimize
nonspecific background staining. One of two primary anti-IL-21
antibodies was applied (E3149 (mouse>human IL-21-CHO,
HH4.9.1C2.1A6.1C8, PAS) or E2865 (mouse>human IL-21-CHO,
HH4.3.1.2D1.1C12, PAS), both made in house) followed by a
biotinylated goat anti-mouse antibody (Vector Laboratories,
Burlingame, Calif.). A colored reaction product was generated via a
peroxidase-3'3'-diaminobenzidine reaction (ChemMate peroxidase/DAB
staining kit including methyl green counter stain; CMS/Fisher,
Houston, Tex.). The slides coverslipped and then examined under a
light microscope (Nikon Eclipse E600, Nikon Corporation, Tokyo,
Japan).
[0300] The following cells and tissues were tested: BHK cells
transfected with human IL-21 (positive control), BHK-570 cells,
wild type (negative control), and human normal lung, human lung
with chronic perivascular inflammation, human normal lymph node,
human lymph node with B cell lymphoma, human spleen with
myelofibrosis, and human duodenum. These tissues were obtained on a
contract basis either from CHTN (Nashville, Tenn.) or NDRI
(Philadelphia, Pa.). Also tested were normal human tissues on a
multi-tissue slide (Biomeda, Hayward, Calif.) and human
abnormal/tumor tissues on a multi-tissue slide (Biomeda, Hayward,
Calif.).
[0301] The E3149 antibody produced positive staining only in the
transfected BHK cells. With the E2865 antibody, intense staining
was observed in the positive control cells as well as occasional
mononuclear cells of unknown identity in the epithelium of the
small intestine in the normal multi-tissue block. The location in
the small intestine from which this sample was obtained is unknown.
This positive cell type was rare (1 cell in 3 sections) in a
separate section of human duodenum.
[0302] Moreover, a positive population of mononuclear cells was
diffusely distributed throughout the human spleen from a patient
with myelofibrosis. A similar staining pattern was not found in the
spleens of the normal multi-human tissue block. Although there was
some staining in the spleens of the multi-tissue block, the
staining was not clearly cell associated. In addition, a section of
inflamed lung contained stained spindle-shaped cells and
mononuclear cells in what looks like the subpleural space (the size
and quality of section make determination of location difficult).
Positive staining was observed in scattered pituicytes in
pituitaries in the multitissue block. Isotype antibody stained
pituitaries were negative. Colloid staining was observed in thyroid
sections of the multi-tissue block and in a section of thyroid
adenocarcinoma in the multi-tumor block. The significance of this
is unknown-the colloid in the isotype section occasionally was
stained (but much less intensely than in the corresponding
anti-IL-21 stained tissues). Light staining was also seen in the
thyroid follicular epithelium, but its intensity was near
background levels.
[0303] Staining in the undifferentiated carcinoma in the
multi-tumor block is associated with a central area of necrotic
debris mixed with inflammatory cells-the specificity of this
staining is questionable. Likewise, staining in the pancreatic
adenocarcinoma may be associated with necrotic debris or associated
inflammatory cells.
[0304] Because of the location of staining in the above tissues it
is possible that IL-21 plays a role in intestinal mucosal immunity
(occasional cells in gut epithelium), inflammation (associated with
inflammatory cells in lung, undifferentiated carcinoma, pancreatic
adenocarcinoma) and myelofibrosis; fibrosis in the bone marrow
resulting in extramedullary hematopoiesis in the spleen-could this
also involve proliferation of the lineage of cells that produces
IL-21 with IL-21 attempting to regulate the process. A caveat with
these results is that we get different staining patterns with
different antibodies. This may be due to the recognition of
different epitopes by the two antibodies.
Example 25
IL-21 Promotes IL-2 Stimulated NK-Cell Expansion in PBMNC Cultures
in the Presence of IL-4
[0305] IL-4 inhibits the expansion of NK-cells stimulated with
IL-2. In two experiments human peripheral blood mononuclear cells
(PBMNCs) were seeded at 200,000 cells/well in alpha-MEM+ 10%
autologous serum with 10 ng/ml IL-2 (R&D Systems, Minneapolis,
Minn.) with or without 0.5 ng/ml IL-4 (R&D Systems,
Minneapolis, Minn.), and with or without 10 ng/ml IL-21 (U.S. Pat.
No. 6,307,024) and grown for 8 days. The number of viable cells per
well was determined using standard methods and the cells analyzed
by flow cytometry for expression of CD3, CD16, and CD56. NK-cells
were defined as the CD56 positive CD3 negative population.
[0306] The cultures from the two donors cultured with IL-2 alone
contained about 151,000 and 326,000 NK-cells respectively on day 8.
The cultures from the two donors cultured with IL-2 and IL-21
contained about 446,000 and 588,000 NK-cells respectively. The
cultures from the two donors cultured with IL-2 and IL-4 contained
about 26,000 and 29,000 NK-cells on day 8. However, cultures from
the two donors cultured with IL-2, IL-4 and IL-21 contained about
229,000 and 361,000 NK-cells representing an 8.8 and 12.5 fold
increase in NK-cell yield over the culture with IL-2 and IL-4
only.
[0307] These results demonstrate that IL-21 promotes NK-cell
expansion, and that IL-21 can largely overcome the inhibitory
effects of IL-4 on NK-cell growth. In some diseases IL-4 expression
can play a role in the pathology. For example mice bearing the
B16F10 melanoma generate a large population of IL-4 producing CD4+
T-cells which appear to limit the host anti-tumor response.
Furthermore, STAT 6 (required for IL-4 signaling) gene deficient
mice exhibit an enhanced ability to reject tumors. The ability of
IL-21 to antagonize the action of IL-4, and induce the expression
of IFN-.gamma. (described herein), in addition to the in vivo
anti-tumor activity and data described herein, suggest that IL-21
can be useful in treating malignacies, infections or autoimmune
disease where there is a Th2 response limiting the hosts ability to
control the disease.
Example 26
IL-21 Synerzizes with IL-2 to Promote the Growth of NK Cells from
Peripheral Blood.
[0308] Peripheral blood lymphocytes from a healthy human donor were
prepared by a standard Ficoll centrifugation method. Lymphocytes
were magnetically negatively enriched as described herein using the
human NK cell negative enrichment system from Stem Cell
Technologies. NK's were cultured at a starting concentration of
about 75,000/ml in 2 ml/well alpha MEM with 10% donor serum, 50
.mu.M BME, 2 ng/ml flt3L, and 0, 0.5, 10, or 50 ng/ml of IL-2+/-0,
5, or 50 ng/ml IL-21. After 15 days of culture cells were
harvested, counted, and analyzed by flow cytometry for CD3, CD56,
and CD161. All cells analyzed after 15 days of culture were
CD3-/CD56+, which are defined as NK cells. At day 0, cells were
analyzed by flow cytometry and found to be >98% CD3-/CD56+.
[0309] The "fold increase" in cell number is defined as the final
cell number divided by the starting cell number. Any "fold
increase" below 1 is therefore a decrease in cell number. In
general, the results at 10 ng/ml of IL-2 were similar to the
results obtained with 50 ng/ml of IL-2, and the results at 5 ng/ml
IL-21 were similar to those obtained with 50 ng/ml IL-21. With no
IL-2 present, the fold increase in total cells was 0.064. When 5
ng/ml IL-21 was included in the culture, the fold increases was
0.11. This indicates that IL-21 has very little proliferative
activity on NK's by itself. At a low concentration of 0.5 ng/ml
IL-2, we saw a fold increase of 0.25. When 5 ng/ml IL-21 was
included, we saw a fold increase of 2.9. At higher concentrations
of IL-2 the fold increases were overall higher, but the effect of
IL-21 was in general decreased, although still positive. At 10
ng/ml IL-2 we saw a fold increase of 2.9. When 5 ng/ml IL-21 was
included, we saw a fold increase of 7.
[0310] IL-21's effect in these cultures was dependent on the
presence of at least low dose IL-2. Without IL-2, the effect of
IL-21 was minimal. When IL-2 is present, especially at the lower,
perhaps physiological, concentration, IL-21's effect is the most
profound. The lack of effect of IL-21 alone, coupled with its
ability to synergize with low concentrations of other cytokines may
allow it to act therapeutically at sites of infection or malignancy
without causing systemic toxicity.
Example 27
IL-21 Stimulates Outgrowth of NK and NKT From Peripheral Blood
Lymphocyte Cultures that Contain IL-2 or IL-15
[0311] Peripheral blood lymphocytes from 3 healthy human donors
were prepared by the standard Ficoll centrifugation method.
Lymphocytes were then cultured at a starting concentration of
200,000/ml in alpha MEM with 10% donor serum, 50 .mu.M BME (Sigma),
2 ng/ml flt3L (R&D Systems), and 0, 0.5, 10, or 50 ng/ml of
IL-2 (R&D Systems) or IL-15 (R&D Systems) +/-0, 5, or 50
ng/ml IL-21 (U.S. Pat. No. 6,307,024). After 12 days of culture
cells were harvested, counted, and analyzed by flow cytometry for
CD3, CD56, and CD8. NK cells were defined as CD56+/CD3- and NKT
cells were defined as CD56+/CD3+.
[0312] The "fold increase" in cell number (defined as final cell
number/starting cell number) was widely variable between the three
donors, but the trends were fairly consistent. In general, the
results at 10 ng/ml of IL-2 or IL-15 were similar to the results
obtained with 50 ng/ml of IL-2 or IL-15, and the results at 5 ng/ml
IL-21 were similar to those obtained with 50 ng/ml IL-21. With no
IL-2 or IL-15 present, the fold increase in total cells was 0.33,
0.23, and 0.19 among the three donors. When 5 ng/ml IL-21 was
included in the culture, the fold increases were 0.47, 0.31, and
0.35. At a low concentration of 0.5 ng/ml IL-2, we saw total cell
fold increases of 2.2, 1.1, and 1.0 among the three donors. When 5
ng/ml IL-21 was included, we saw total cells increase by 5.5, 2.3,
3.1. We saw fold increases in NK numbers without IL-21 of 16, 4.2,
and 3.5. When IL-21 was present (at 5 ng/ml) these increases were
24, 15, and 21 respectively. NKT's were also positively effected
under these conditions. NKT fold increases were 4.4, 5.7, and 1.8
without IL-21, and 10, 9, and 15 with 5 ng/ml IL-21.
[0313] These results are mirrored with IL-15. At 0.5 ng/ml IL-15, a
total cell fold increases of 0.98, 0.43, and 0.88 was seen among
the three donors. When 5 ng/ml IL-21 was included, we saw total
cells increase by 1.4, 0.9, 1.7 fold. Fold increases of NK numbers
of 8.0, 0.85, and 3.7 were seen without IL-21. When 5 ng/ml IL-21
was present, these fold increases were 13, 5.5, and 11. NKT fold
increases at 0.5 ng/ml IL-15 were 3.3, 2.3, and 1.6 for the three
donors, but they were 3.9, 5.2, and 4.7 when 5 ng/ml IL-21 was
included.
[0314] At higher concentrations of IL-2 the fold increases were
overall higher, but the effect of IL-21 was in general decreased,
although still positive. At 10 ng/ml IL-2 we saw total cell fold
increases of 18, 2.5, and 2.8 among the three donors. When 5 ng/ml
IL-21 was included, we saw total cells increase by 21, 3.6, 9.8
fold. We saw fold increases in NK numbers of 114, 13, and 13
without IL-21. When IL-21 was also present (at 5 ng/ml) these
increases were 100, 19, and 56 respectively. NKT's were also
positively effected under these conditions. NKT fold increases were
33, 15, and 12 without IL-21, and 52, 20, and 38 with 5 ng/ml
IL-21.
[0315] At 10 ng/ml IL-15, we saw total cell fold increases of 18,
0.8, and 1.7 among the three donors. When 5 ng/ml IL-21 was
included, we saw total cells increase by 23, 1.4, 6.9 fold. We saw
fold increases of NK numbers of 128, 0.58, and 2.0 without IL-21.
When 5 ng/ml IL-21 was present, these fold increases were 107, 1.1,
and 9.4. NKT fold increases at 10 ng/ml IL-15 were 60, 6.5, and 5.7
for the three donors, but they were 66, 12, and 33 when 5 ng/ml
IL-21 was included.
[0316] IL-21's effects in these cultures were dependent on the
presence of at least low dose IL-2 or IL-15. Without those
cytokines, the effect of IL-21 was minimal. When IL-2 or IL-15 is
present, especially at the lower, perhaps physiological,
concentrations, IL-21's effect is the most profound. The lack of
effect of IL-21 alone, coupled with its ability to synergize with
low concentrations of other cytokines may allow it to act
therapeutically at sites of infection or malignancy without causing
systemic toxicity.
Example 28
IL-21 Inhibits the Production of IL-13 in NK-Cell Cultures.
[0317] IL-13 shares receptor subunits and many of the biological
activities of IL-4, but unlike IL-4, IL-13 is produced by NK-cells.
Since NK-cells also produce IFN-.gamma., and these two cytokines
have in large part opposing activities, experiments were conducted
to examine the effects of IL-21 on IL-13 and IFN-.gamma. expression
in PBMNC and NK-cell cultures.
[0318] Negatively selected human peripheral blood NK-cells were
seeded at about 3.75.times.10e5 cells/ml and stimulated 2days with
10 ng/ml IL-2, IL-4 (R&D Systems) or IL-21 (U.S. Pat. No.
6,307,024) or without any cytokine in alpha-MEM+10% autologous
serum. After two days in culture IL-2 was added to all wells to 10
ng/ml, and the cell were cultured for an additional 3 days, then
the supernatants were collected and analyzed by ELISA for IL-13 and
IFN-.gamma.. NK-cells grown for two days without any cytokine
produced about 2130 pg/ml IFN-.gamma. and 175 pg/ml IL-13. Cells
stimulated with IL-21 produced abut 10,300 pg/ml IFN-.gamma. and 90
pg/ml IL-13. Cells stimulated with IL-2 produced 12,700 pg/ml
IFN-.gamma. and 1000 pg/ml IL-13. Cells stimulated with IL-4
produced no detectable IFN-.gamma. nor IL-13.
[0319] Of note, cells stimulated for the first two days with IL-21
produced 5 times more IFN-.gamma., but only one half the IL-13 of
unstimulated cells. When compared to cell stimulated with IL-2 for
the first two days of culture cells stimulated with IL-21 produced
80% as much IFN-.gamma., but only 9% as much IL-13. Thus IL-21
selectively promotes the expression of IFN-.gamma. and depresses
IL-13 expression.
Example 29
IL-21 Synergizes with IL-2 to Promote IFN-.gamma. Production in
Mouse Splenic NK Cells
[0320] C57BL/6 mouse splenic NK cells were prepared by water lysing
a cell suspension from the spleens, then utilizing the Stem Cell
Technologies murine NK negative enrichment magnetic cell sorting
protocol. The cells prepared using this method were 65% Pan NK
positive based on flow analysis using the DX5 Pan NK antibody from
PharMingen.
[0321] The negatively enriched murine NK cells were cultured for 8
days at 500,000 cells/ml in RPMI 1640 with 10% heat inactivated
fetal bovine serum and 2 mM L-glutamine, 50 .mu.M BME, and PSN
antibiotic with 20 ng/ml mIL-2 (R&D Systems) or 10 ng/ml mIL-21
(U.S. Pat. No. 6,307,024) or both. The cell supernatants were
harvested and cells were counted at the end of the culture period.
Cell supernatants were assayed for mIFN-.gamma. using a
commercially available ELISA kit from PharMingen.
[0322] Cell numbers at the end of the eight day period were about
1,300,000 for the IL-2 containing culture, 220,000 for the
IL-2/IL-21 culture, and 10,000 for the IL-21 culture. The
mIFN-.gamma. levels were 2.2 ng/ml, 30 ng/ml, and 0.28 ng/ml
respectively. When expressed as pg/500,000 cells, the results are
238, 14,000, and 12,000. IL-21 enhances IFN-.gamma. expression in
these cultures, which when combined with the cell
survival/proliferation effects of IL-2, results in high levels of
IFN-.gamma. being secreted into the medium. IFN-.gamma. is an
important initiator of the immune response, and is considered a TH1
biased cytokine. This data supports that IL-21 plays a role in the
anti-cancer, antiviral activity of the immune system, and hence can
be used as a therapeutic in anti-cancer, anti-viral and other
applications.
Example 30
Effects IL-21-Saporin Toxin Conjugate on T Cells and Human T Cell
Lines
[0323] The ability of the murine IL-21-saporin toxin conjugate to
bind normal murine T Cells was determined by FACS competition
assays and compared to binding on the same cells by the murine
IL-21. It was shown that the murine IL-21-saporin toxin conjugate
binds to these cells with the same affinity as IL-21 (Example
30A).
[0324] The presence of Human IL-21 Receptor (WIPO Publication No.s
WO 0/17235 and WO 01/77171) on the following T Cell lines was
determined by FACS analysis: human T Cell leukemia MOLT-13 (DSMZ
No. ACC.sub.--436), human cutaneous T Cell lymphoma HUT-78 (ATCC
No. TIB.sub.--161), human cutaneous T Cell lymphoma HUT-102 (ATCC
No. TIB.sub.--162); human ALCL line DEL (DSMZ No. ACC.sub.--338),
and human T/NK cell leukemia YT (DSMZ No. ACC.sub.--434; Example
30B).
[0325] The effects of human IL-21-saporin toxin conjugate described
herein were tested on both normal human T Cells (Example 30C) and
the Human T cell lines shown to express the Human IL-21 Receptor
(i.e., MOLT-13, HUT-78, HUT-102, DEL and YT; example 30D). The
results showed that normal Human T cells and T cell lines treated
with IL-21-saporin toxin conjugate proliferated much less or not at
all as compared with cells left untreated or cells cultured with
unconjugated IL-21 or with saporin alone.
[0326] The results indicate that IL-21-toxin conjugate (saporin or
other) can control some types of T-cell neoplasms with little or no
effect also on normal Human T cell proliferation in vitro at 30 pM
or less. A proposed mechanism of the observed inhibition of cell
line proliferation in vitro is as follows: the Human IL-21-toxin
conjugate binds with high affinity to IL-21 receptor expressed on
the surface of these cells. The Human IL-21-toxin conjugate is then
taken up by the cells and, in the case with the saporin-toxin
conjugate, the cells' ability to produce protein and their
proliferation is subsequently blocked. Thus, IL-21-saporin
immunotoxin conjugate, or other IL-21-toxin fusion could be used
therapeutically in prevention and treatment T-cell leukemias and
lymphomas, and other cancers wherein IL-21 receptors are
expressed.
A. The Binding of Murine IL-21-Salorin Toxin Conjugate on Normal
Murine T Cells by Flow Cytometry Analysis.
[0327] Total murine splenocytes were isolated from normal 4-month
old female C57/BL6 mice (Jackson Laboratories, Bar Harbor, Me.).
Spleens were harvested and gently mashed between frosted slides to
create a cell suspension. Red blood cells were removed by hypotonic
lysis as follows: cells were pelleted and the supernatant removed
by aspiration. We disrupted the pellet with gentle vortexing, then
added 900 ul of sterile water while shaking, followed quickly (less
than 5 sec later) by 100 ul of 10.times.HBSS (Gibco/BRL; Rockville
Md.). The cells were then resuspended in 10 ml of 1.times.HBSS and
debris was removed by passing the cells over a nylon mesh-lined
cell strainer (Falcon/BD; Franklin N.J.). These RBC-depleted spleen
cells were then pelleted and resuspended in FACS stain buffer: HBSS
(GibcoBRL; Rockville Md.) containing 3% Human serum, 1% BSA, and 10
mM HEPES.
[0328] Aliquots containing about 1.times.10.sup.6 white blood cells
from spleen were stained for 3-color flow cytometric analysis with
anti-murine CD3-FITC, anti-murine B220-CyChrome mAbs (PharMingen,
San Diego, Calif.) and biotinylated murine IL-21 (2 ug/ml) followed
by Streptavidin-PE (Caltag; Burlingame Calif.). Staining of the
biotinylated murine IL-21 was competed by equivalent titered molar
amounts (0.0175 nM to 3.5 nM) of both unbiotinylated IL-21 and
IL-21-saporin conjugate. Cells were analyzed on a FACSScan using
CellQuest software (Becton Dickinson, Mountain View, Calif.). The
results demonstrated that the murine IL-21-saporin toxin conjugate
binds to these cells with the same affinity as IL-21.
B. The Binding of Biotinylated Murine IL-21 on Human T Cell Lines
by Flow Cytometry Analysis.
[0329] Aliquots containing 0.4.times.10 exp6 to 1.times.10 exp6
MOLT-13 cells, HuT-78 cells, HuT-102 cells, DEL cells or YT cells
were stained for 1-color flow cytometric analysis with titered
biotinylated murine IL-21 (40 ng/ml to 1000 ng/ml) followed by
Streptavidin-PE (Caltag; Burlingame Calif.). Cells were analyzed on
a FACSScan using CellQuest software (Becton Dickinson, Mountain
View, Calif.). The results demonstrated that biotinylated murine
IL-21 binds to these cell lines with strong affinity. The results
also demonstrated that the murine IL-21 is cross-species reactive
as it recognizes and binds the Human IL-21 Receptor molecule on the
surface of the Human T cell lines.
C. The Effect of Human IL-21-Saporin Immunotoxin on Normal Human
T-cell Proliferation.
[0330] Whole blood was collected from a healthy human donor,
aliquoted into 50 ml tubes and passed over Ficoll density
gradients. RBC-depleted cells at the interface (PBMC) were
collected and washed extensively with PBS followed by RPMI 1640
supplemented with 10% human ultraserum and 2 mM L glutamine. The
PBMC were suspended to 111.times.10exp6/ml in MACS buffer (PBS, 1%
BSA, 0.8 mg/L EDTA). Cells were combined with anti-human CD14
microbeads, anti-human CD19 microbeads, and anti-human CD56
microbeads (Miltenyi Biotech; Auburn Calif.) as per manufacturer
specifications. The mixture was incubated for 20 min. at 4.degree.
C. These cells labeled with CD14, CD19 and CD56 beads were washed
with 5.times. volume MACS buffer, and then resuspended in 1.5 ml
MACS buffer.
[0331] A VS+ column (Miltenyi Biotech; Auburn Calif.) was prepared
according to the manufacturer's instructions. The VS+ column was
then placed in a VarioMACS.TM. magnetic field (Miltenyi Biotech;
Auburn Calif.). The column was equilibrated with 3 ml MACS buffer.
The CD14/CD19/CD56 bead-coated PBMC were then applied to the
column. The CD14-CD19-CD56- PBMC fraction containing Human T cells
were allowed to pass through the column and were collected in a
15ml tube. The column was washed with I Oml (2.times.5 ml) MACS
buffer to wash out residual Human T Cells. The column with bound
CD14+CD19+CD56+ cells was discarded. The Human T cells and wash
eluant were pooled together and counted.
[0332] A sample of the negatively-selected human T cells was
removed for staining to assess the fraction's purity. A
cychrome-conjugated mouse anti-human CD3 antibody (PharMingen) was
used for staining the selected cells. The negatively-selected T
cells were shown to be 67% CD3+.
[0333] The isolated primary T cells were cultured at
0.5.times.10.sup.6/ml with equivalent titered molar amounts (0.03
pM to 30 pM) of both IL-21 and IL-21-saporin conjugate on a plate
coated with titered (0 to 2 .mu.g/ml) anti-human CD3 (clone UCHT-1;
Southern Biotech; Birmingham Ala.) as a co-activator for the Human
T cells. After 3 days growth, the cells were pulsed with
3H-thymidine (5 .mu.Ci/ml; AmershamBiosciences, Piscataway N.J.)
for 18 hours. Cells were lysed and DNA was captured onto glass
filter mats (Packard, Meriden Conn.) and counted for 3H-thymidine
incorporation to assess proliferation of the cells.
[0334] The results showed that IL-21 at 0.3 pM and higher had a
stimulatory effect in combination with 2 .mu.g/ml coated anti-CD3.
The IL-21-saporin conjugate had no such stimulatory effect. It also
had no inhibitory effect as compared with the 2 tg/ml anti-CD3 coat
stimulus alone. In sum, these data can be interpreted to mean that
while a IL-21 stimulus was present in the wells containing the
IL-21-saporin conjugate, the expected increase in a proliferation
due to the IL-21 part of the molecule was ablated by the presence
of the saporin portion of the conjugated molecule. However, the
saporin portion of the molecule did not ablate the stimulatory
effect due to the anti-CD3 coated antibody.
D. The Effect of Human IL-21-Saporin Immunotoxin Conjugate on Human
T Cell Lines.
[0335] Cells were seeded at 5,000 cells/ml to 50,000 cells/ml with
equivalent titered molar amounts (0.2 pM to 400 pM) of both IL-21
and IL-21-saporin conjugate. After 2 days growth the cells were,
pulsed with 5 .mu.Ci/ml .sup.3H-thymidine (AmershamBiosciences,
Piscataway N.J.) for 18 hours. Cells were either lysed and DNA was
captured onto glass filter mats (Packard, Meriden Conn.) and
counted for .sup.3H-thymidine incorporation to assess proliferation
of the cells.
[0336] IL-21-saporin treated MOLT-13 cells had incorporated
.sup.3H-thymidine to only 70% of the .sup.3H-thymidine
incorporation by the IL-21 treated MOLT-13 cells. IL-21-saporin
treated HuT-78 cells grew to only 33% the density of the untreated
HuT-78 cells. IL-21-saporin treated HuT-102 cells grew to only 20%
the density of the untreated Hut- 102 cells. IL-21-saporin treated
DEL cells grew to only 25% the density of the untreated DEL cells.
IL-21 -saporin treated YT cells grew to only 33% the density of the
untreated YT cells. The results indicate that IL-21-toxin conjugate
(saporin or other) can be effective in controlling some types of
T-cell neoplasms. Moreover, IL-21-saporin immunotoxin conjugate, or
other IL-21-toxin fusion could be used therapeutically in
prevention and treatment T-cell leukemias and lymphomas, and other
cancers wherein IL-21 receptors are expressed.
Example 31
In vivo Effects of IL-21 on B-cell Lymphomas
[0337] Human B-lymphoma cell lines are maintained in vitro by
passage in growth medium. The cells are washed thoroughly in PBS to
remove culture components.
[0338] SCID Mice are injected with (typically) one million human
lymphoma cells via the tail vein in a 100 microliter volume. (The
optimal number of cell injected is determined empirically in a
pilot study to yield tumor take consistently with desired
kinetics.) IL-21 treatment is begun the next day by either
subcutaneous. implantation of an ALZET.RTM. osmotic mini-pump
(ALZET, Cupertino, Calif.) or by daily i.p injection of IL-21 or
vehicle. Mice are monitored for survival and significant morbidity.
Mice that lose greater than 20% of their initial body weight are
sacrificed, as well as mice that exhibit substantial morbidity such
as hind limb paralysis. Depending on the lymphoma cell line
employed, the untreated mice typically die in 3 to 6 weeks. For B
cell lymphomas that secrete IgG or IgM, the disease progression can
also be monitored by weekly blood sampling and measuring serum
human Immunoglobulin levels by ELISA.
A. IL-21 Dose Response/IM-9 Model
[0339] Mice were injected with 1.times.10.sup.6 IM-9 cells, and 28
day osmotic mini pumps implanted the following day. The pumps were
loaded with the following concentrations of IL-21 to deliver: 0,
0.12, 1.2 or 12 micrograms per day with 8 mice per dose group.
IL-21 exhibited a clear dose dependent effect in protecting mice
from the tumor cell line. The effects of IL-21 were dose dependent.
Surviving mice at the end of the experiment had no signs of disease
and no detectable human IgG in their serum.
B. IL-21 NK Depletion/IM-9 Model
[0340] Mice were depleted of NK-cells by administering 5 doses of
anti-asialo-GM-1 antibody every third day beginning 15 days prior
to injection of tumor cells or left undepleted as controls. Half of
the depleted and undepleted mice were treated with 12 .mu.g/day
IL-21 and the other half were treated with vehicle only. Depletion
of NK-cells did not significantly diminish the activity of IL-21.
These data demonstrated that NK-cells are not necessary for the
effect of IL-21 in the IM-9 model in SCID mice.
C. Other Cell Lines Tested
[0341] The following additional cell lines were tested using the
model shown for IM-9 cells. IL-21 delivered at 12 .mu.g/day by
minipump is effective against CESS cells in SCID mice. IL-21
administered to mice with RAJI cell implanted tumors had no
efficacy. IL-21 administered to mice with RAMOS cell implanted
tumors had no efficacy. IL-21 administered to mice with HS SULTAN
cell implanted tumors had significant efficacy, but did not prevent
disease in most mice, only slows its onset. IL-21 DoHH2 had no
efficacy.
[0342] These data demonstrate that the efficacy of IL-21 in SCID
mouse lymphoma models correlates with the ability to inhibit the
growth of the lymphoma cell lines in vivo. Furthermore, NK-cell
depletion of SCID mice for both T-cells and B-cells does not
diminish the effectiveness of IL-21 in the IM-9 model. It is likely
that the efficacy of IL-21 in SCID mouse lymphoma models is
dependent on it direct effects on the tumor cells because no
efficacy was seen in three of three cell lines tested in the model
that were not inhibited by IL-21 in vitro, and NK depletion had no
effect on the efficacy of IL-21 in the IM-9 model. In a patient
with an intact immune system, IL-21 dependent effector cell
mediated antitumor effects are predicted from experiments with
immunocompetent mice in syngeneic tumor models. The demonstration
of direct antitumor effects in SCID mice suggests that IL-21
therapy could have combined direct and effector mediated anti-tumor
effects in selected B-cell malignancies in humans.
Example 32
[0343] The Effects of IL-21 in a Mouse Syngeneic Ovarian Carcinoma
Model
[0344] The effect of IL-21 is tested for efficacy in ovarian
carcinoma using a mouse syngeneic model as described in Zhang et
al., Am. J. of Pathol. 161:2295-2309, 2002. Briefly, using
retroviral transfection and fluorescence-activated cell sorting a
C57BL6 murine ID8 ovarian carcinoma cell line is generated that
stably overexpresses the murine VEGF164 isoform and the enhanced
green fluorescence protein (GFP). The retroviral construct
containing VEGF164 and GFP cDNAs was transfected into BOSC23 cells.
The cells are analyzed by FACS cell sorting and GFP high positive
cells are identified.
[0345] The ID8 VEGF164/GFP transfected cells are cultured to
subconfluence and prepared in a single-cell suspension in phosphate
buffer saline (PBS) and cold MATRIGEL (BD Biosciences, Bedford,
Mass.). Six to eight week old femal C57BL6 mice are injected
subcutaneously in the flank at 5.times.10.sup.6 cells or
untransfected control cells. Alternatively, the mice can be
injected intraperitoneally at 7.times.10.sup.6 cells or control
cells. Animals are either followed for survival or sacrificed eight
weeks after inoculation and evaluated for tumor growth. Mice are
treated with recombinant. murine IL-21 beginning 3-14 days
following tumor implantation, or when tumor engraftment and growth
rate is established. Treatment levels of 0.5-5 mg/kg will be
administered on a daily basis for 5-14 days, and may be continued
thereafter if no evidence of neutralizing antibody formation is
seen.
Example 33
The Effects of IL-21 in a Mouse RENCA Model
[0346] The efficacy of IL-21 in a renal cell carcinoma model is
evaluated using BALB/c mice that have been injected with RENCA
cells, a mouse renal adenocarcinoma of spontaneous origin,
essentially as described in Wigginton et al., J. Nat. Cancer
Instit. 88:38-43, 1996.
[0347] Briefly, BALB/c mice between eight and ten weeks are
injected with RENCA cells R 1.times.10.sup.5 cells into the kidney
capsule of the mice. Twelve days after tumor cell implantation, the
mice are nepharectomized to remove primary tumors. The mice are
allowed to recover from surgery, prior to administration of IL-21.
Mice are treated with recombinant. murine IL-21 beginning 3-14 days
following tumor implantation, or when tumor engraftment and growth
rate is established. Treatment levels of 0.5-5 mg/kg will be
administered on a daily basis for 5-14 days, and may be continued
thereafter if no evidence of neutralizing antibody formation is
seen. Alternatively, RENCA cells may be introduced by subcutaneous
(5.times.10e5 cells) or intravenous (1.times.10.sup.5 cells)
injection,
[0348] The mice are evaluated for tumor response as compared to
untreated mice. Survival is compared using a Kaplan-Meier method,
as well as tumor volume being evaluated.
Example 34
The Effects of IL-21 in a Mouse Colorectal Tumor Model
[0349] The effects of IL-21 in a colorectal mouse model are tested
as described in Yao et al., Cancer Res. 63:586-592, 2003. In this
model, MC-26 mouse colon tumor cells are implanted into the splenic
subcapsul of BALB/c mice. After 14 days, the treated mice are
administered IL-21. Mice are treated with recombinant. murine IL-21
beginning 3-14 days following tumor implantation, or when tumor
engraftment and growth rate is established. Treatment levels of
0.5-5 mg/kg will be administered on a daily basis for 5-14 days,
and may be continued thereafter if no evidence of neutralizing
antibody formation is seen.
[0350] The efficacy of IL-21 in prolonging survival or promoting a
tumor response is evaluated using standard techniques described
herein.
Example 35
The Effect of IL-21 in a Mouse Pancreatic Cancer Model
[0351] The efficacy of IL-21 in a mouse pancreatic cancer model is
evaluated using the protocol developed by Mukherjee et al., J.
Immunol. 165:3451-3460, 2000. Briefly, MUC1 transgenic (MUC1.Tg)
mice are bred with oncogene-expressing mice that spontaneously
develop tumors of the pancreas (ET mice) designated as MET. MUC1.Tg
mice. ET mice express the first 127 aa of SV40 large T Ag under the
control of the rat elastase promoter. Fifty percent of the animals
develop life-threatening pancreatic tumors by about 21 wk of age.
Cells are routinely tested by flow cytometry for the presence of
MUC 1. All mice are on the C57BL/6 background. Animals are
sacrificed and characterized at 3-wk intervals from 3 to 24 wk.
Mice are carefully observed for signs of ill-health, including
lethargy, abdominal distention, failure to eat or drink, marked
weight loss, pale feces, and hunched posture.
[0352] The entire pancreas is dissected free of fat and lymph
nodes, weighed, and spread on bibulus paper for photography.
Nodules are counted, and the pancreas is fixed in methacarn,
processed for microscopy by conventional methods, step sectioned at
5 .mu.m (about 10 sections per mouse pancreas), stained with
hematoxylin and eosin, and examined by light microscopy. Tumors are
obtained from MET mice at various time points during tumor
progression, fixed in methacarn (60% methanol, 30% chloroform, 10%
glacial acetic acid), embedded in paraffin, and sectioned for
immunohistochemical analysis. MUC1 antibodies used are CT1, a
rabbit polyclonal Ab that recognizes mouse and human cytoplasmic
tail region of MUCI, HMFG-2, BC2, and SM-3, which have epitopes in
the TR domain of MUC1.
[0353] Determination of CTL activity is performed using a standard
.sup.51Cr release method after a 6-day in vitro peptide stimulation
without additional added cytokines. Splenocytes from individual MET
mice are harvested by passing through a nylon mesh followedby lysis
of RBC.
[0354] Single cells from spleens of MET mice are analyzed by
two-color immunofluorescence for alterations in lymphocyte
subpopulations: CD3, CD4, CD8, Fas, FasL, CD11c, and MHC class I
and II. Intracellular cytokine levels were determined after cells
are stimulated with MUC1 peptide (10 .mu.g/ml for 6 days) and
treated with brefeldin-A (also called Golgi-Stop; PharMingen) as
directed by the manufacturer's recommendation (4
.mu.l/1.2.times.10.sup.7 cells/6 ml for 3 h at 37.degree. C. before
staining). Cells are permeabilized using the PharMingen
permeabilization kit and stained for intracellular IFN-.gamma.,
IL-2, IL-4, and IL-5 as described by PharMingen. All fluorescently
labeled Abs were purchased from PharMingen. Flow cytometric
analysis was done on Becton Dickinson FACscan using the CellQuest
program (Becton Dickinson, Mountain View, Calif.).
[0355] Mice are treated with recombinant. murine IL-21 beginning
3-14 days following tumor implantation, or when tumor engraftment
and growth rate is established. Treatment levels of 0.5-5 mg/kg
will be administered on a daily basis for 5-14 days, and may be
continued thereafter if no evidence of neutralizing antibody
formation is seen.
Example 36
The Effects of IL-21 in a Murine Breast Cancer Model
[0356] The efficacy of IL-21 in a murine model for breast cancer is
made using a syngeneic model as described in Colombo et al., Cancer
Research 62:941-946, 2002. Briefly, TS/A cells which are a
spontaneous mammary carcinoma for BALB/C mice. The cells are
cultured for approximately one week to select for clones. The
selected TS/A cells are grown and used to challenge CD-I nu/nu BR
mice (Charles River Laboratories) by injected 2.times.10.sup.2 TS/A
cells subcutaneously into the flank of the mouse.
[0357] Mice are treated with recombinant. murine IL-21 beginning
3-14 days following tumor implantation, or when tumor engraftment
and growth rate is established. Treatment levels of 0.5-5 mg/kg
will be administered on a daily basis for 5-14 days, and may be
continued thereafter if no evidence of neutralizing antibody
formation is seen. The tumors are excised after sacrificing the
animals and analyzed for volume and using histochemistry and
immunohistochemistry.
Example 37
The Effects of IL-21 in a Murine Prostate Cancer Model
[0358] The effects of IL-21 on tumor response are evaluated in
murine prostate cancer model, using a model similar to that
described in Kwon et al., PNAS 96:15074-15079, 1999. In this model,
there is a metastatic outgrowth of transgenic adenocarcinoma of
mouse prostate (TRAMP) derived prostate cancer cell line TRAMP-C2,
which are implanted in C57BL/6 mice. Metastatic relapse is
reliable, occurring primarily in the draining lymph nodes in close
proximity to the primary tumor.
[0359] Briefly, the C2 cell line used is an early passage line
derived from the TRAMP mouse that spontaneously develops
autochthonous tumors attributable to prostate-restricted SV40
antigen expression. The cells are cultured and injected
subcutaneously into the C57BL/6 mice at 2.5-5.times.10.sup.6
cells/0.1 ml media. Mice are treated with recombinant. murine IL-21
beginning 3-14 days following tumor implantation, or when tumor
engraftment and growth rate is established. Treatment levels of
0.5-5 mg/kg will be administered on a daily basis for 5-14 days,
and may be continued thereafter if no evidence of neutralizing
antibody formation is seen. The tumors are excised after
sacrificing the animals and analyzed for volume and using
histochemistry and immunohistochemistry.
Example 38
The Effects of IL-21 and Chemotherapeutics on Growth of Human
B-Cell Lines In Vitro
[0360] The effects of IL-21 and zeocin alone and in combination
were tested on the growth of IM-9 and HS Sultan human B-cell lines
in vitro to ascertain if the growth inhibitory/cytotoxic effects of
IL-21 and a chemo-therapeutic agent will be additive or synergistic
on IL-21 sensitive cells lines. ZEOCIN ( Invitrogen, Carlsbad,
Calif.) is an antibiotic with a mechanism of action similar to the
related chemotherapeutic bleomycin was used.
[0361] IM-9 and HS Sultan cell lines were plated at 50,000 cells/ml
in RPMI1640 medium suppplemented with 2 mM L-glutamine and 10% heat
inactivated FBS with and without IL-21 (at 20 ng/ml) and or ZEOCIN
(at 15.6 .mu.g/ml for IM-9s and 31 tg/ml for HS Sultans) for two
days then the cells were harvested and washed once to remove the
zeocin and replated with or without IL-21 in a serial cell dilution
series, with 6 wells per dilution, in 96 well round bottom plates
to determine their relative growth ability. The plates were scored
in 6 days using Alamar blue, as a measure of viable cells per well,
and reflecting their survival and outgrowth from the prior
treatment. Cells populations treated with ZEOCIN had less than one
tenth the growth capacity of untreated cells, and the combination
of IL-21 with zeocin further reduced the growth capacity by
approximately an order of magnitude. These data suggest that IL-21
could be successfully combined with chemotherapy to augment
response rates in the treatment of lymphoma.
[0362] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
17 1 642 DNA Homo sapiens CDS (47)...(532) 1 gctgaagtga aaacgagacc
aaggtctagc tctactgttg gtactt atg aga tcc 55 Met Arg Ser 1 agt cct
ggc aac atg gag agg att gtc atc tgt ctg atg gtc atc ttc 103 Ser Pro
Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe 5 10 15 ttg
ggg aca ctg gtc cac aaa tca agc tcc caa ggt caa gat cgc cac 151 Leu
Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His 20 25
30 35 atg att aga atg cgt caa ctt ata gat att gtt gat cag ctg aaa
aat 199 Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys
Asn 40 45 50 tat gtg aat gac ttg gtc cct gaa ttt ctg cca gct cca
gaa gat gta 247 Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro
Glu Asp Val 55 60 65 gag aca aac tgt gag tgg tca gct ttt tcc tgt
ttt cag aag gcc caa 295 Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys
Phe Gln Lys Ala Gln 70 75 80 cta aag tca gca aat aca gga aac aat
gaa agg ata atc aat gta tca 343 Leu Lys Ser Ala Asn Thr Gly Asn Asn
Glu Arg Ile Ile Asn Val Ser 85 90 95 att aaa aag ctg aag agg aaa
cca cct tcc aca aat gca ggg aga aga 391 Ile Lys Lys Leu Lys Arg Lys
Pro Pro Ser Thr Asn Ala Gly Arg Arg 100 105 110 115 cag aaa cac aga
cta aca tgc cct tca tgt gat tct tat gag aaa aaa 439 Gln Lys His Arg
Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys 120 125 130 cca ccc
aaa gaa ttc cta gaa aga ttc aaa tca ctt ctc caa aag atg 487 Pro Pro
Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met 135 140 145
att cat cag cat ctg tcc tct aga aca cac gga agt gaa gat tcc 532 Ile
His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser 150 155 160
tgaggatcta acttgcagtt ggacactatg ttacatactc taatatagta gtgaaagtca
592 tttctttgta ttccaagtgg aggagcccta ttaaattata taaagaaata 642 2
162 PRT Homo sapiens 2 Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile
Val Ile Cys Leu Met 1 5 10 15 Val Ile Phe Leu Gly Thr Leu Val His
Lys Ser Ser Ser Gln Gly Gln 20 25 30 Asp Arg His Met Ile Arg Met
Arg Gln Leu Ile Asp Ile Val Asp Gln 35 40 45 Leu Lys Asn Tyr Val
Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro 50 55 60 Glu Asp Val
Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln 65 70 75 80 Lys
Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile 85 90
95 Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala
100 105 110 Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp
Ser Tyr 115 120 125 Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe
Lys Ser Leu Leu 130 135 140 Gln Lys Met Ile His Gln His Leu Ser Ser
Arg Thr His Gly Ser Glu 145 150 155 160 Asp Ser 3 3072 DNA Mus
musculus CDS (54)...(491) 3 gagaaccaga ccaaggccct gtcatcagct
cctggagact cagttctggt ggc atg 56 Met 1 gag agg acc ctt gtc tgt ctg
gta gtc atc ttc ttg ggg aca gtg gcc 104 Glu Arg Thr Leu Val Cys Leu
Val Val Ile Phe Leu Gly Thr Val Ala 5 10 15 cat aaa tca agc ccc caa
ggg cca gat cgc ctc ctg att aga ctt cgt 152 His Lys Ser Ser Pro Gln
Gly Pro Asp Arg Leu Leu Ile Arg Leu Arg 20 25 30 cac ctt att gac
att gtt gaa cag ctg aaa atc tat gaa aat gac ttg 200 His Leu Ile Asp
Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp Leu 35 40 45 gat cct
gaa ctt cta tca gct cca caa gat gta aag ggg cac tgt gag 248 Asp Pro
Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys Glu 50 55 60 65
cat gca gct ttt gcc tgt ttt cag aag gcc aaa ctc aag cca tca aac 296
His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser Asn 70
75 80 cct gga aac aat aag aca ttc atc att gac ctc gtg gcc cag ctc
agg 344 Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu
Arg 85 90 95 agg agg ctg cct gcc agg agg gga gga aag aaa cag aag
cac ata gct 392 Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys
His Ile Ala 100 105 110 aaa tgc cct tcc tgt gat tcg tat gag aaa agg
aca ccc aaa gaa ttc 440 Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg
Thr Pro Lys Glu Phe 115 120 125 cta gaa aga cta aaa tgg ctc ctt caa
aag atg att cat cag cat ctc 488 Leu Glu Arg Leu Lys Trp Leu Leu Gln
Lys Met Ile His Gln His Leu 130 135 140 145 tcc tagaacacat
aggacccgaa gattcctgag gatccgagaa gattcccgag 541 Ser gactgaggag
acgccggaca ctatagacgc tcacgaatgc aggagtacat cttgcctctt 601
gggattgcaa gtggagaagt acgatacgtt atgataagaa caactcagaa aagctatagg
661 ttaagatcct ttcgcccatt aactaagcag acattgtggt tccctgcaca
gactccatgc 721 tgtcaacatg gaaaatctca actcaacaag agcccagctt
cccgtgtcag ggatttctgg 781 tgcttctcaa gctgtggctt catcttattg
cccaactgtg acattctttg attggaaggg 841 gaaaactaaa gcttttagca
aaaatacagc tagggaattt gtcgatctgc gagagtaaga 901 cctcttatga
tcctaacgga atgatgtaag ctggaaataa taagcataag atgaaattga 961
aaattgaagt ctttattctt taagaaaaac tttgtacttg aaagcatgtc tgaagagttt
1021 actcattacc acaaacatct agcatattga taactaacat ctttatactc
tacaagagag 1081 gctttccaga taggtacagt ttttcttctc tattaggtct
atcaaaattt aacctattat 1141 gagggtcacc cctggctttc actgtttttc
taaagaggca agggtgtagt aagaagcagg 1201 cttaagttgc cttcctccca
atgtcaagtt cctttataag ctaatagttt aatcttgtga 1261 agatggcaat
gaaagcctgt ggaagtgcaa acctcactat cttctggagc caagtagaat 1321
tttcaagttt gtagctctca cctcaagtgg ttatgggtgt cctgtgatga atctgctagc
1381 tccagcctca gtctcctctc ccacatcctt tcctttcttt cctctttgaa
acttctaaga 1441 aaaagcaatc caaacaagtt cagcacttaa gacacattgc
atgcacactt ttgataagtt 1501 aaatccaacc atctatttaa aatcaaaatc
aggagatgag ccaagagacc agaggttctg 1561 ttccagtttt aaacagactt
ttactgaaca tcccaatctt ttaaccacag aggctaaatt 1621 gagcaaatag
ttttgccatt tgatataatt tccaacagta tgtttcaatg tcaagttaaa 1681
aagtctacaa agctattttc cctggagtgg tatcatcgct ttgagaattt cttatggtta
1741 aaatggatct gagatccaag catggcctgg gggatggttt tgatctaagg
aaaaaggtgt 1801 ctgtacctca cagtgccttt aaaacaagca gagatcccgt
gtaccgccct aagatagcac 1861 agactagtgt taactgattc ccagaaaagt
gtcacaatca gaaccaacgc attctcttaa 1921 actttaaaaa tatgtattgc
aaagaacttg tgtaactgta aatgtgtgac tgttgatgac 1981 attatacaca
catagcccac gtaagtgtcc aatggtgcta gcattggttg ctgagtttgc 2041
tgctcgaaag ctgaagcaga gatgcagtcc ttcacaaagc aatgatggac agagagggga
2101 gtctccatgt tttattcttt tgttgtttct ggctgtgtaa ctgttgactt
cttgacattg 2161 tgatttttat atttaagaca atgtatttat tttggtgtgt
ttattgttct agccttttaa 2221 atcactgaca atttctaatc aagaagtaca
aataattcaa tgcagcacag gctaagagct 2281 tgtatcgttt ggaaaagcca
gtgaaggctt ctccactagc catgggaaag ctacgcttta 2341 gagtaaacta
gacaaaattg cacagcagtc ttgaacctct ctgtgctcaa gactcagcca 2401
gtcctttgac attattgttc actgtgggtg ggaacacatt ggacctgaca cactgttgtg
2461 tgtccatgaa ggttgccact ggtgtaagct ttttttggtt ttcattctct
tatctgtaga 2521 acaagaatgt ggggctttcc taagtctatt ctgtatttta
ttctgaactt cgtatgtctg 2581 agttttaatg ttttgagtac tcttacagga
acacctgacc acacttttga gttaaatttt 2641 atcccaagtg tgatatttag
ttgttcaaaa agggaaggga tatacataca tacatacata 2701 catacataca
tatatatata tatatataca tatatatata tatatatatg tatatatata 2761
tatatataga gagagagaga gagagagaga gagaaagaga gagaggttgt tgtaggtcat
2821 aggagttcag aggaaatcag ttatggccgt taatactgta gctgaaagtg
ttttctttgt 2881 gaataaattc atagcattat tgatctatgt tattgctctg
ttttatttac agtcacacct 2941 gagaatttag ttttaatatg aatgatgtac
tttataactt aatgattatt tattatgtat 3001 ttggttttga atgtttgtgt
tcatggcttc ttatttaaga cctgatcata ttaaatgcta 3061 cccagtccgg a 3072
4 146 PRT Mus musculus 4 Met Glu Arg Thr Leu Val Cys Leu Val Val
Ile Phe Leu Gly Thr Val 1 5 10 15 Ala His Lys Ser Ser Pro Gln Gly
Pro Asp Arg Leu Leu Ile Arg Leu 20 25 30 Arg His Leu Ile Asp Ile
Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp 35 40 45 Leu Asp Pro Glu
Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys 50 55 60 Glu His
Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser 65 70 75 80
Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu 85
90 95 Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His
Ile 100 105 110 Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr
Pro Lys Glu 115 120 125 Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys
Met Ile His Gln His 130 135 140 Leu Ser 145 5 1614 DNA Homo sapiens
CDS (1)...(1614) 5 atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg
ctg ctc cag gga 48 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu
Leu Leu Gln Gly 1 5 10 15 ggc tgg ggc tgc ccc gac ctc gtc tgc tac
acc gat tac ctc cag acg 96 Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr
Thr Asp Tyr Leu Gln Thr 20 25 30 gtc atc tgc atc ctg gaa atg tgg
aac ctc cac ccc agc acg ctc acc 144 Val Ile Cys Ile Leu Glu Met Trp
Asn Leu His Pro Ser Thr Leu Thr 35 40 45 ctt acc tgg caa gac cag
tat gaa gag ctg aag gac gag gcc acc tcc 192 Leu Thr Trp Gln Asp Gln
Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60 tgc agc ctc cac
agg tcg gcc cac aat gcc acg cat gcc acc tac acc 240 Cys Ser Leu His
Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr 65 70 75 80 tgc cac
atg gat gta ttc cac ttc atg gcc gac gac att ttc agt gtc 288 Cys His
Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95
aac atc aca gac cag tct ggc aac tac tcc cag gag tgt ggc agc ttt 336
Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100
105 110 ctc ctg gct gag agc atc aag ccg gct ccc cct ttc aac gtg act
gtg 384 Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr
Val 115 120 125 acc ttc tca gga cag tat aat atc tcc tgg cgc tca gat
tac gaa gac 432 Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp
Tyr Glu Asp 130 135 140 cct gcc ttc tac atg ctg aag ggc aag ctt cag
tat gag ctg cag tac 480 Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln
Tyr Glu Leu Gln Tyr 145 150 155 160 agg aac cgg gga gac ccc tgg gct
gtg agt ccg agg aga aag ctg atc 528 Arg Asn Arg Gly Asp Pro Trp Ala
Val Ser Pro Arg Arg Lys Leu Ile 165 170 175 tca gtg gac tca aga agt
gtc tcc ctc ctc ccc ctg gag ttc cgc aaa 576 Ser Val Asp Ser Arg Ser
Val Ser Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190 gac tcg agc tat
gag ctg cag gtg cgg gca ggg ccc atg cct ggc tcc 624 Asp Ser Ser Tyr
Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205 tcc tac
cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt cag 672 Ser Tyr
Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220
acc cag tca gag gag tta aag gaa ggc tgg aac cct cac ctg ctg ctt 720
Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu 225
230 235 240 ctc ctc ctg ctt gtc ata gtc ttc att cct gcc ttc tgg agc
ctg aag 768 Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser
Leu Lys 245 250 255 acc cat cca ttg tgg agg cta tgg aag aag ata tgg
gcc gtc ccc agc 816 Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp
Ala Val Pro Ser 260 265 270 cct gag cgg ttc ttc atg ccc ctg tac aag
ggc tgc agc gga gac ttc 864 Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys
Gly Cys Ser Gly Asp Phe 275 280 285 aag aaa tgg gtg ggt gca ccc ttc
act ggc tcc agc ctg gag ctg gga 912 Lys Lys Trp Val Gly Ala Pro Phe
Thr Gly Ser Ser Leu Glu Leu Gly 290 295 300 ccc tgg agc cca gag gtg
ccc tcc acc ctg gag gtg tac agc tgc cac 960 Pro Trp Ser Pro Glu Val
Pro Ser Thr Leu Glu Val Tyr Ser Cys His 305 310 315 320 cca cca cgg
agc ccg gcc aag agg ctg cag ctc acg gag cta caa gaa 1008 Pro Pro
Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu 325 330 335
cca gca gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc ttc tgg
1056 Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe
Trp 340 345 350 ccg aca gcc cag aac tcg ggg ggc tca gct tac agt gag
gag agg gat 1104 Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser
Glu Glu Arg Asp 355 360 365 cgg cca tac ggc ctg gtg tcc att gac aca
gtg act gtg cta gat gca 1152 Arg Pro Tyr Gly Leu Val Ser Ile Asp
Thr Val Thr Val Leu Asp Ala 370 375 380 gag ggg cca tgc acc tgg ccc
tgc agc tgt gag gat gac ggc tac cca 1200 Glu Gly Pro Cys Thr Trp
Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro 385 390 395 400 gcc ctg gac
ctg gat gct ggc ctg gag ccc agc cca ggc cta gag gac 1248 Ala Leu
Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp 405 410 415
cca ctc ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt gtc tca
1296 Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys Val
Ser 420 425 430 gct ggc agc cct ggg cta gga ggg ccc ctg gga agc ctc
ctg gac aga 1344 Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser
Leu Leu Asp Arg 435 440 445 cta aag cca ccc ctt gca gat ggg gag gac
tgg gct ggg gga ctg ccc 1392 Leu Lys Pro Pro Leu Ala Asp Gly Glu
Asp Trp Ala Gly Gly Leu Pro 450 455 460 tgg ggt ggc cgg tca cct gga
ggg gtc tca gag agt gag gcg ggc tca 1440 Trp Gly Gly Arg Ser Pro
Gly Gly Val Ser Glu Ser Glu Ala Gly Ser 465 470 475 480 ccc ctg gcc
ggc ctg gat atg gac acg ttt gac agt ggc ttt gtg ggc 1488 Pro Leu
Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly 485 490 495
tct gac tgc agc agc cct gtg gag tgt gac ttc acc agc ccc ggg gac
1536 Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly
Asp 500 505 510 gaa gga ccc ccc cgg agc tac ctc cgc cag tgg gtg gtc
att cct ccg 1584 Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val
Val Ile Pro Pro 515 520 525 cca ctt tcg agc cct gga ccc cag gcc agc
1614 Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 530 535 6 538 PRT Homo
sapiens 6 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu
Gln Gly 1 5 10 15 Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp
Tyr Leu Gln Thr 20 25 30 Val Ile Cys Ile Leu Glu Met Trp Asn Leu
His Pro Ser Thr Leu Thr 35 40 45 Leu Thr Trp Gln Asp Gln Tyr Glu
Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60 Cys Ser Leu His Arg Ser
Ala His Asn Ala Thr His Ala Thr Tyr Thr 65 70 75 80 Cys His Met Asp
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95 Asn Ile
Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105 110
Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val 115
120 125 Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu
Asp 130 135 140 Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu
Leu Gln Tyr 145 150 155 160 Arg Asn Arg Gly Asp Pro Trp Ala Val Ser
Pro Arg Arg Lys Leu Ile 165 170 175 Ser Val Asp Ser Arg Ser Val Ser
Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190 Asp Ser Ser Tyr Glu Leu
Gln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205 Ser Tyr Gln Gly
Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220 Thr Gln
Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu 225 230 235
240 Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys
245
250 255 Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro
Ser 260 265 270 Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser
Gly Asp Phe 275 280 285 Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser
Ser Leu Glu Leu Gly 290 295 300 Pro Trp Ser Pro Glu Val Pro Ser Thr
Leu Glu Val Tyr Ser Cys His 305 310 315 320 Pro Pro Arg Ser Pro Ala
Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu 325 330 335 Pro Ala Glu Leu
Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp 340 345 350 Pro Thr
Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp 355 360 365
Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala 370
375 380 Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr
Pro 385 390 395 400 Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro
Gly Leu Glu Asp 405 410 415 Pro Leu Leu Asp Ala Gly Thr Thr Val Leu
Ser Cys Gly Cys Val Ser 420 425 430 Ala Gly Ser Pro Gly Leu Gly Gly
Pro Leu Gly Ser Leu Leu Asp Arg 435 440 445 Leu Lys Pro Pro Leu Ala
Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro 450 455 460 Trp Gly Gly Arg
Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser 465 470 475 480 Pro
Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly 485 490
495 Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp
500 505 510 Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile
Pro Pro 515 520 525 Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 530 535
7 153 PRT Homo sapiens 7 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile
Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser
Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu
Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met
Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80
Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85
90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val
Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys
Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn
Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr
145 150 8 153 PRT Homo sapiens 8 Met Gly Leu Thr Ser Gln Leu Leu
Pro Pro Leu Phe Phe Leu Leu Ala 1 5 10 15 Cys Ala Gly Asn Phe Val
His Gly His Lys Cys Asp Ile Thr Leu Gln 20 25 30 Glu Ile Ile Lys
Thr Leu Asn Ser Leu Thr Glu Gln Lys Thr Leu Cys 35 40 45 Thr Glu
Leu Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr 50 55 60
Glu Lys Glu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr 65
70 75 80 Ser His His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala
Gln Gln 85 90 95 Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys
Arg Leu Asp Arg 100 105 110 Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser
Cys Pro Val Lys Glu Ala 115 120 125 Asn Gln Ser Thr Leu Glu Asn Phe
Leu Glu Arg Leu Lys Thr Ile Met 130 135 140 Arg Glu Lys Tyr Ser Lys
Cys Ser Ser 145 150 9 162 PRT Homo sapiens 9 Met Arg Ile Ser Lys
Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu
Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val
Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40
45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp
Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser
Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn
Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys
Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu
Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr
Ser 10 144 PRT Homo sapiens 10 Met Trp Leu Gln Ser Leu Leu Leu Leu
Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro
Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln
Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala
Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp
Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70
75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr
Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro
Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe
Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp
Cys Trp Glu Pro Val Gln Glu 130 135 140 11 22 DNA Artificial
Sequence Oligonucleotide primer ZC22281 11 tgtgaatgac ttggtccctg aa
22 12 23 DNA Artificial Sequence Oligonucleotide primer ZC22279 12
aacaggaaaa agctgaccac tca 23 13 31 DNA Artificial Sequence zalpha11
Ligand TaqMan probe, ZG32 13 tctgccagct ccagaagatg tagagacaaa c 31
14 20 DNA Artificial Sequence Oligonucleotide primer ZC22277 14
ccaggagtgt ggcagctttc 20 15 21 DNA Artificial Sequence
Oligonucleotide primer ZC22276 15 gcttgccctt cagcatgtag a 21 16 23
DNA Artificial Sequence zalpha11 TaqMan(r) probe, designated ZG31
16 cggctccccc tttcaacgtg act 23 17 8 PRT Artificial Sequence
OVA257-264 peptide 17 Ser Ile Ile Asn Phe Glu Lys Leu 1 5
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