Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF MODULATING MEMORY EFFECTOR T-CELLS AND
COMPOSITIONS
TECHNICAL FIELD OF THE INVENTION
This invention relates to methods of using inhibitors of the CD2/LFA-3
interaction
in treating conditions characterized by the presence of activated T cells in
mammals,
including humans. Such conditions include inflammatory bowel diseases,
psoriatic
arthritis, rheumatoid arthritis, and multiple sclerosis.
~o BACKGROUND OF THE INVENTION
Antigen presenting cells (APC) ~ ~:press a high density of Class ll major
histocompatibility complex (MHC) antigen on the cell surface. MHC Class B
molecules
bind peptides derived from endocytosed antigen and are recognized primarily by
helper T
lymphocytes. The T cell receptor on T cells recognizes antigen as a peptide
fragment
~5 bound to the cell-surface molecules encoded by the MHC (Springer, "Adhesion
Receptors
of the Immune System", Nature, 346, pp. 425-27 ( 1990)).
There are many interactions between molecules expressed on the surface of such
APC and the surface of T cells, in addition to the T cell receptor/MHC
interaction. These
surface molecules, often referred to as adhesion molecules, participate in a
number of
20 functions including cellular adhesion, antigen recognition,
co-stimulatory signaling in T cell activation and stimulation of effectors of
T cell
cytotoxicity ("Adhesion Molecules in Diagnosis and Treatment of Inflammatory
Diseases",
The Lancet, 336, pp. 1351-52 (1990)). Such cell adhesion appears to be
involved in
activation of T cell proliferation in the generation of an immune response
(Hughes et al.,
25 "The Endothelial Cell as a Regulator of T-cell Function", Immunol. Rev.,
117, pp. 85-102
( 1990)).
One way in which T cells are activated is by binding of their antigen specific
T cell
receptors to peptide-MHC complexes on the surface of antigen presenting cells
such as
macrophages. T cell activation stimulates proliferation and differentiation of
two types of
3o functional T cells: helper cells, which promote the proliferation and
maturation of antibody
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producing B lymphocytes; and killer cells, which lyse target cells (Bierer et
al., "A
Monoclonal Antibody to LFA-3, the CD2 Ligand, Specifically Immobilizes Major
Histocompatibility Complex Proteins", Eur. J. Immunol. 19: pp. 661-65 (1989);
Springer
"Adhesion Receptors of the Immune System", Nature, 346, pp: 425-34 (1990)).
There are numerous medical conditions characterized by abnormal immune
responses and many of these are, in particular, characterized by increased T
cell activation.
T cells play a major role in the immune response by interacting with target
and antigen
presenting cells. For example, T cell-mediated killing of target cells is a
multi-step process
involving, initially, adhesion of cytolytic T cells (the effector cells) to
target cells. Also,
to helper T cells help initiate the immune response by adhesion to antigen
presenting cells.
These interactions of T cells with target and antigen presenting cells are
highly
specific and depend on the recognition of an antigen on the surface of a
target or APC by
one of the many specific antigen receptors on the surface of T cells.
The receptor-antigen interaction of T cells and other cells is also
facilitated by
various T cell surface proteins, e.g., the antigen-receptor complex CD3 and
accessory
adhesion molecules such as CD4, LFA-1, CDB, and CD2. It is also facilitated by
accessory
adhesion molecules, such as LFA-3, ICAM- I and MHC, that are expressed on the
surface
of the target or antigen presenting cells. For example, LFA- I and its counter
receptor
ICAM- I or ICAM-2, as well as CD2 and its counter receptor LFA-3 have been
implicated
in cellular adhesion and T cell activation. It is known that the LFA-1/ICAM
and
CD2/LFA-3 interactions are independent. A number of other molecules present on
resting
T cells have also been implicated in T cell adhesion, including E2 (MIC2), VLA-
4
(CD49d), CD44 (Hermes, Pgp-1, ECMRIII), and H19 (N4) (see Makgoba et al., "The
CD2-LFA-3 and LFA-1-ICAM Pathways: Relevance to T-cell Recognition", Immunol,
Today, 10, pp. 417-22 ( 1989)).
The interaction between CD2 and LFA-3 remains poorly understood with respect
to
activation of T cell activity. Recent studies have suggested that there is a
specific
interaction between CD2 (a T cell adhesion molecule) and LFA-3 (a target cell
and APC
adhesion molecule) which mediates T cell adhesion to the target or antigen
presenting
3o cells. This cell-cell adhesion has been implicated in the initiation of T
cell functional
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responses (Dustin et-al., "Purified Lymphocyte Function Associated Antigen 3
Binds to
CD2 and Mediates T Lymphocyte Adhesion." J. Ex-p. Mcd., 165, pp. 677-92
(1987);
Springer et al., "The Lymphocyte Function-associated LFA-I, CD2, and LFA-3
Molecules:
Cell Adhesion Receptors of the Immune System", Ann. Rev. Immunol., 5, pp. 223-
52
s ( 1987)).
LFA-3, which is found on the surface of a wide variety of cells, including
human
erythrocytes, has become the subject of a considerable amount of study to
further elucidate
its role in various T cell interactions (see, e.g., Krensky et al., "The
Functional
Significance, Distribution, and Structure of LFA-1. LFA-2, and LFA-3: Cell
Surface
t0 Antigen Associated with CTL-Target Interactions". J. Immunol., 131(2), pp.
611-16
( 1983); Shaw et al., "Two Antigen-Independent Adhesion Pathways Used by Human
Cytotoxic T-cell Clones", Nature, 323, pp. 262-64 ( 1986)).
Two natural forms of LFA-3 have been identified. One form of LFA-3
("transmembrane LFA-3") is anchored in the cell membrane by a transmembrane
i5 hydrophobic domain. cDNA encoding this form of LFA-3 has been cloned and
sequenced
(see, e.g., Wallner et al., "Primary Structure of Lymphocyte Function-
Associated Antigen-3
(LFA-3)", J. Ex.,p. Med., 166, pp. 923-32 (1987)). Another form of LFA-3 is
anchored to
the cell membrane via a covalent linkage to phosphatidylinositol ("PI")-
containing
glycolipid. This latter form has been designated "PI-linked LFA-3", and CDNA
encoding
20 this form of LFA-3 has also been cloned and sequenced (Wallner et al., PCT
Pub. WO
90/02181).
The human CD2 molecule is a 50 kD surface glycoprotein expressed on >95% of
thymocytes and virtually all peripheral T lymphocytes. Biochemical analyses
using
specific monoclonal antibodies have suggested that CD2 is T lineage-specific
and exists on
25 the cell surface in several differentially glycosylated forms (Howard et
al., "A Human T
Lymphocyte Differentiation Marker Defined by Monoclonal Antibodies that Block
E-
Rosette Formation", J. Immunol., 126, pp. ? 117-22 ( 198 1 ); Brown et aL, in
Leukocyte
Typin~, ed. McMichael, Oxford University Press, pp. I 10-12 (1987); Sayre et
al.,
"Molecular Cloning and Expression of T11 eDNAs Reveals a Receptor-Like
Structure on
30 Human T Lymphocytes", Proc, Natl. Acad. Sci. USA, 84, pp. 2941-45 (1987)).
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The sequence of a human CD2 gene has been reported (Seed and Aruffo,
"Molecular Cloning of the CD2 Antigen, the T-cell Erythrocyte Receptor, by a
Rapid
Immunoselection Procedure". Proc. Natl. Acad. Sci, USA, 84, pp. 3365-b9 (
1987); Sayre
et al., "Molecular Cloning and Expression of TI 1 cDNAs Reveal a Receptor-like
Structure
on Human T Lymphocytes", Proc. Natl. Acad, Sci, USA, 84, pp. 2941-45 (1987)).
CD2
cDNA clones predict a cleaved signal peptide of 24 amino acid residues, an
extracellular
segment of 185 residues, a transmembrane domain of 25 residues and a
cytoplasmic region
of 117 residues (Sayre et al., supra C 1987); Sewell et al., "Molecular
Cloning of the
Human T-Lymphocyte Surface CD2 (Tl 1 ) Antigen", Proc. Natl. Acad. Sci. USA,
83, pp.
8718-32 ( 1986); Seed and Aruffo, supra ( 1987); Clayton et al., Eur. J.
lmmun., 17, pp.
1367-70 (1987)). Soluble CD2 polypeptides having an LFA-3 binding domain have
been
reported (PCT Publn. WO 90/08187). Monoclonal antibodies to CD2, for example
TS2/18, T11,, T11~, T1 I,, and to LFA3, for example TS2/9, have also been
reported (see,
e.g., Hughes et al., "Tbe Endothelial Cell as a Regulator of T-Cell Function".
Immunol.
t_5 Reviews, I 17, pp. 85-I02 (1990); Meuer, "An Alternative Pathway of T-Cell
Activation: A
Functional Role for the 50 kD TI 1 Sheep Erythrocyte Receptor Protein", Cell,
36, pp. 897-
906 ( 1984)).
Binding of the antigen specific T cell receptors to peptide-MHC complexes on
the
surface of antigen presenting cells is also involved in the formation of so-
called "memory"
2o cells which are generally formed during the adaptive immune response
requiring contact
with antigen and expansion of antigen-specific memory cells. T-cell memory is
probably
dependent upon repeated stimulation by antigen. Memory T-cells augment their
binding
avidity for the antigen-presenting cell through increased expression of CD2,
LFA-3 and
other accessory adhesion molecules. Indeed, an important phenotypic change in
the
25 isoform of the leukocyte antigen CD45R allows a distinction to be made
between naive T
cells which express CD45R and memory T-cells which react with the lower
molecular
weight form CD45R0.
The realization that most, if not all, memory cells are subject to repeated
bombardment from antigen makes it likely that most of the features associated
with the
3o CD45R0 subset are in fact manifestations of effector T cells. There is a
growing and
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provocative literature describing the association of CD45R0 memory effector T
cells with
the presence of various inflammatory diseases such as inflammatory bowel
diseases,
including Crohn's disease and ulcerative colitis, (Horiuchi et al., J. Japan.
Soc. Colo.
Proctol., 45: 391-393 (1992)); psoriatic arthritis (Veale et al., Ann. Rheum.
Dis. 53: 450-
s 454 (1994); rheumatoid arthritis (Muller et al., Autoimmunity 14: 37-43
(1992); uveitis
(Ohta et al., Curr. Eye Res., 15: 299-306 (1996), nephritis (Rodruguez-Poerez
et al., Am. J.
Nephrol., 15: 386-391 (1995); and multiple sclerosis ( Crucian et al., Clin.
Dia~Lab.
Immunol., 2: 249-252 ( 1995), Lehmann et al., Clin. Immunol. Immunovathol.,
85: 202-
209 (L997), Muller et al., Autoimmunitv 14: 37-43 (1992), Qin et al., J. Clin.
Immunot., I3:
~0 152-161 (1993).
Because of the possible role of these memory effector T lymphocytes in
pathogenesis, the need exists for improved methods of modulating these cells
in patients
with these conditions.
~5 SUMMARY OF THE INVENTION
The present invention provides a method of preventing or treating conditions
characterized by infiltration of memory effector T lymphocytes in an organ of
the
mammal. In the methods, a CD2 binding agent is administered that is capable of
modulating the number and/or distribution of memory effector T lymphocytes.
The
20 methods of this invention are superior to previously available therapies
for these
conditions for many reasons, including the fact that the methods are more
selective, thus
less immunosuppressive than pre-existing therapies with less general toxicity.
Materials that can be used in accordance with the method of the present
invention
include any molecule that is a CD2 binding agent. This includes molecules or
other
25 chemical entities that modulate the CD2/LFA-3 interaction. Preferably, the
agent is
selected from the group consisting of anti-LFA-3 antibody homologs, anti-CD2
antibody
homologs, soluble LFA-3 polypeptides, soluble CD2 polypeptides, CD2 or LFA-3
mimetic
agents (such as small organic molecules) and derivatives thereof.
One aspect of the invention is a method for selectively modulating memory
effector
3o T lymphocytes in a subject having a medical condition, the method
comprising
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administering to the subject an effective amount of a CD2
binding agent. The memory T lymphocytes may infiltrate a
target organ in the subject. Preferably, the memory
effector T lymphocytes are CD45R0 T lymphocytes. The CD2
binding agent is selected from the group consistng of
anti-LFA-3 antibody homologs, anti-CD2 antibody homologs,
soluble LFA-3 polypeptides, soluble CD2 polypeptides, CD2
mimetic agents, and LFA-3 mimetic agents. Another method of
the invention involves treating a condition in a subject
characterized by implication of memory ~ffector
T lymphocytes in pathogenesis, the method including
administering to the subject an effective amount of a CD2
binding agent. The condition is selected from the group
consisting of psoriatic arthritis, rheumatoid arthritis,
multiple sclerosis, atopic dermatitis, uveitis, inflammatory
bowel disease, Crohn's disease, ulcerative colitis, and
cutaneous T cell lymphoma.
A further method of the invention is a method of
modulating memory effector T lymphocytes in a mammal,
wherein the mammal is characterized by having the presence
of infiltrating memory effector T lymphocytes in an organ of
the mammal. The method comprises administering to the
mammal an amount of a CD2 binding agent sufficient to
selectively modulate memory effector T lymphocytes in said
organ. The mammal suffers from a condition selected from
the group consisting of psoriatic arthritis, rheumatoid
arthritis, multiple sclerosis, atopic dermatitis, uveitis,
inflammatory bowel disease, Crohn's disease, ulcerative
colitis, and cutaneous T cell lymphoma.
Compositions of the invention comprise a
population of memory effector T lymphocytes obtainable from
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a mammal having a condition characterized by the presence of
infiltrating memory effector T lymphocytes in an organ of
the mammal, wherein the population is in combination with a
CD2 binding agent.
In one aspect, the invention provides use of a
composition comprising an effective amount of a CD2-binding
LFA-3 polypeptide for treating a subject having a medical
condition selected from the group consisting of: psoriatic
arthritis, uveitis, inflammatory bowel disease, Crohn's
l0 disease, and ulcerative colitis.
In another aspect, the invention provides use of a
composition comprising an effective amount of a CD2-binding
LFA-3 polypeptide fox treating a subject having rheumatoid
arthritis, wherein the LFA-3 polypeptide is used in
combination with methotrexate.
In another aspect, the invention provides use of a
composition comprising an effective amount of a CD2-binding
LFA-3 polypeptide for treating a subject having rheumatoid
arthritis, wherein the LFA-3 polypeptide is used in
combination with cyclosporin A.
In another aspect, the invention provides use of a
composition comprising an effective amount of a CD2-binding
LFA-3 polypeptide for treating a subject having psoriatic
arthritis, wherein the LFA-3 polypeptide is used in
combination with methotrexate.
In another aspect, the invention provides use of a
composition comprising an effective amount of a CD2-binding
LFA-3 polypeptide for treating a subject having psoriatic
arthritis, wherein the LFA-3 polypeptide is used in
combination with cyclosporin A.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing the selective
reduction of memory effector T cells relative to naive T
cells in patients with psoriasis receiving treatment with
the CD2 binding agent, LFA3-TIP. The horizontal bar above
the X axis represents the duration of treatment, which was
stopped at approximately 80 days. The curves represent the
mean lymphocyte counts from the 57 patients in each
treatment group. There are statistically significant
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.7.
treatment and temporal effects with regard to the memory T cells (CD 45 RO+)
but not
with regard to the naive T cells (CD45 RA+). T cell counts in the various CD2
binding
agent treatments show a decrease, approaching some relatively constant value,
during the
80 day adminstration period. After treatment was terminated, the memory
effector T cells
count began to return.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, "effector T lymphocyte", "effector T cells", ''memory effector
T
0 lymphocyte/cell" and similar terms are used interchangeably and mean T
lymphocytes that
nave already been exposed to antigen (i.e., those that are not ''naive"). More
specifically,
the terms mean CD45R0 T lymphocytes, as opposed to "naive" CD45RA T
lymphocytes.
As used herein, "CD2" means a CD2 polypeptide that binds to a naturally
occurring '
LFA-3 polypeptide and which is encoded by (a) a naturally occurring mammalian
CD2
t5 DNA sequence (e.g., SEQ ~ NO:S); (b) a DNA sequence degenerate to a
naturally
occurring CD2 DNA sequence; or (c) a DNA sequence that hybridizes to one of
the
foregoing DNA sequences under conditions equivalent to about 20 degrees C to
27 degrees
C below Tm and IM sodium chloride.
As used herein, " LFA-3 " means an LFA-3 polypeptide that binds to a naturally
20 occurring CD2 polypeptide and which is encoded by (a) a naturally occurring
mammalian
LFA-3 DNA sequence (e.g., SEQ )D NO: 1 or SEQ m N0:3); (b) a DNA sequence
degenerate to a naturally occurring LFA-3 DNA sequence; or (c) a DNA sequence
that
hybridizes to one of the foregoing DNA sequences under standard hybridization
conditions,
as defined below.
25 As used herein, a "CD2 binding agent" is a molecule, or compound, or other
chemical entity that modulates the CD2/LFA3 interaction and/or modulates CD2
signaling.
The term includes anti-LFA-3 antibody homologs, anti-CD2 antibody homologs,
soluble
LFA-3 polypeptides, soluble CD2 polypeptides, CD2 or LFA-3 mimetic agents and
derivatives thereof. The term "modulates" means that the CD2 binding agent
alters
30 (preferably decreases) the intensity of CD2 signaling and/or interferes
with the ability of
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_b_
CD2 to bind to LFA3, although increases in such intensity and/or ability to
bind are not
excluded from the definition. CD? binding agents also "modulate" memory
effector T
lymphocytes (i.e., CD45R0) and in this context the term "modulate" means that
the
binding agents will alter the number and/or distribution of memory effector T
lymphocytes
in a subject receiving the CD? binding agent(s). In preferred methods, the
binding agents)
will "selectively" modulate such memory effector T lymphocytes, meaning that
the
memory effector T lymphocytes are modulated as compared to nave T lymphocytes
(e.g.,
CD45RA T lymphocytes).
As used herein, a "soluble LFA-3 polypeptide" or a "soluble CD2. polypeptide"
is an
LFA-3 or CD2 polypeptide incapable of anchoring itself in a membrane. Such
soluble
polypeptides include, for example, CD2 and LFA-3 polypeptides that lack a
sufficient
portion of their membrane spanning domain to anchor the polypeptide or are
modified such
that the membrane spanning domain is non-functional. As used herein soluble
LFA-3
polypeptides include full-length or truncated (e.g., with internal deletions)
PI-linked LFA-
3.
As used herein, an "antibody homolog" is a protein comprising one or more
polypeptides selected from immunoglobulin light chains, immunoglobulin heavy
chains
and antigen-binding fragments thereof which are capable of binding to one or
more
antigens. The component polypeptides of an antibody homolog composed of more
than
one polypeptide may optionally be disulfide-bound or otherwise covalently
crosslinked.
Accordingly, antibody homologs include intact immunoglobulins of types IgA,
IgG, IgE,
IgD, IgM (as well as subtypes thereof), wherein the light chains of the
immunoglobulin
may be of types kappa or lambda. Antibody homologs also include portions of
intact
immunoglobulins that retain antigen-binding specificity, for example, Fab
fragments, Fab'
fragments, F(ab')2 fragments, F(v) fragments, heavy chain monomers or dimers,
light chain
monomers or dimers, dimers consisting of one heavy and one light chain, and
the like.
As used herein, a "humanized recombinant antibody homolog" is an antibody
homolog, produced by recombinant DNA technology, in which some or all of the
amino
acids of a human immunoglobulin light or heavy chain that are not required for
antigen
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_y_
binding have been substituted for the corresponding amino acids from a non
human
mammalian immunoglobulin light or heavy chain.
As used herein, a "chimeric recombinant antibody homolog" is an antibody
homolog,
produced by recombinant DNA technology, in which all or part of the hinge and
constant
regions of an inu-nunoglobulin light chain, heavy chain, or both, have been
substituted for
the corresponding regions from another immunoglobulin light chain or heavy
chain.
The methods of the invention may be practiced on any mammal, preferably on
humans.
"amino acid"- a monomeric unit of a peptide, polypeptide, or protein. There
are
twenty amino acids found in naturally occurring peptides, polypeptides and
proteins, all of
0 which are L-isomers. The term also includes analogs of the amino acids and D-
isomers of
the protein amino acids and their analogs.
"protein"- any polymer consisting essentially of any of the 20 amino acids.
Although "polypeptidc" is often used in reference to relatively large
polypeptides, and
"peptide" is often used in reference to small polypeptides, usage of these
terms in the art
15 overlaps and is varied. The term "protein" as used herein refers to
peptides, proteins and
polypeptides, unless otherwise noted.
"fusion"- refers to a co-linear linkage of two or more proteins or fragments
thereof via
their individual peptide backbones through genetic expression of a
polynucleotide
molecule encoding those proteins or through protein synthesis methods. It is
preferred that
2o the proteins or fragments thereof be from different sources. Thus,
preferred fusion proteins
include an CD2 binding agent that is a protein or fragment covalently linked
to a second
moiety that is either a different CD2 binding agent or that is not a CD2
binding agent at all.
Specifically, a "CD2 binding agent protein/ Ig fusion" is a protein comprising
a CD2
binding agent of the invention, or fragment thereof linked to an N-terminus of
an
25 immunoglobulin chain wherein a portion of the N-terminus of the
immunoglobulin is
replaced with the CD2 binding agent.
"mutant" - any change in the genetic material of an organism, in particular
any
change (i.e., deletion, substitution, addition, or alteration) in a wild-type
polynucleotide
sequence or any change in a wild-type protein.
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"standard hybridization conditions"- salt and temperature conditions
substantially
equivalent to 0.5-X SSC to about 5 X SSC and 65 ° C for both
hybridization and wash. The
term "standard hybridization conditions" as used herein is therefore an
operational
definition and encompasses a range of hybridization conditions. Higher
stringency
conditions may, for example, include hybridizing with plaque screen buffer
(0.2%
polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serum albumin, 50 mM Tris-
HCl (pH
7.5); 1 M NaCI; 0.1 % sodium pyrophosphate; 1 % SDS); 10% dextran sulphate,
and 100
~tglml denatured, sonicated salmon sperm DNA at 65 ° C for 12-20 hours,
and washing
with 75 mM NaCI/7.5 mM sodium citrate (0.5 x SSC)/1 % SDS at 65° C.
Lower stringency
l0 conditions may, for example, include hybridizing with plaque screen buffer,
10% dextran
sulphate and 110 p.g/ml denatured, sonicated salmon sperm DNA at 55 ° C
for 12-20
hours, and washing with 300 mM NaCI/30mM sodium citrate (2.0 X SSC)/1 % SDS at
55 °
C. See also Current Protocols in Molecular Biology, John Wiley & Sons, lnc.
New
York, Sections 6.3.1-6.3.6, (1989).
~5 "expression control sequence"- a sequence of polynucleotides that controls
and
regulates expression of genes when operatively linked to those genes.
"operatively linked"- a polynucleotide sequence (DNA, RNA) is operatively
linked
to an expression control sequence when the expression control sequence
controls and
regulates the transcription and translation of that polynucleotide sequence.
The term
20 "operatively linked" includes having an appropriate start signal (e.g.,
ATG) in front of the
polynucleotide sequence to be expressed and maintaining the correct reading
frame to
permit expression of the polynucleotide sequence under the control of the
expression
control sequence and production of the desired polypeptide encoded by the
polynucleotide
sequence.
25 "expression vector- a polynucleotide, such as a DNA plasmid or phage (among
other common examples) which allows expression of at least one gene when the
expression
vector is introduced into a host cell. The vector may, or may not, be able to
replicate in a
cell.
"Isolated" (used interchangeably with "substantially pure"j- when applied to
30 nucleic acid i.e., polynucleotide sequences, that encode polypeptides,
means an RNA or
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DNA polynucleotide, portion of genomic polynucleotide, cDNA or synthetic
polynucleotide which, by virtue of its origin or manipulation: (i) is not
associated with all
of a polynucleotide with which it is associated in nature (e.g., is present in
a host cell as an
expression vector, or a portion thereof); or (ii) is linked to a nucleic acid
or other chemical
moiety other than that to which it is linked in nature; or (iii) does not
occur in nature. By
"isolated" it is further meant a polynucleotide sequence that is: (i)
amplified in vitro by, for
example, polymerase chain reaction (PCR); (ii) chemically synthesized; (iii)
recombinantly
produced by cloning; or (iv) purified, as by cleavage and gel separation.
Thus, ''substantially pure nucleic acid" is a nucleic acid which is not
immediately
contiguous with one or both of the coding sequences with which it is normally
contiguous
in the naturally occurring genome of the organism from which the nucleic acid
is derived.
"Isolated" (used interchangeably with "substantially pure")- when applied to
polypeptides means a polypeptide or a portion thereof which, by virtue of its
origin or
manipulation: (i) is present in a host cell as the expression product of a
portion of an
expression vector; or (ii) is linked to a protein or other chemical moiety
other than that to
which it is linked in nature; or (iii) does not occur in nature. By "isolated"
it is further
meant a protein that is : (i) chemically synthesized; or (ii) expressed in a
host cell and
purified away from associated proteins. The term generally means a polypeptide
that has
been separated from other proteins and nucleic acids with which it naturally
occurs.
Preferably, the polypeptide is also separated from substances such as
antibodies or gel
matrices (polyacrylamide) which are used to purify it.
"heterologous promoter"- as used herein is a promoter which is not naturally
associated with a gene or a purified nucleic acid. .
"Homologous"- as used herein is synonymous with the term "identity" and refers
to
the sequence similarity between two polypeptides, molecules or between two
nucleic acids.
When a position in both of the two compared sequences is occupied by the same
base or
amino acid monomer subunit (for instance, if a position in each of the two DNA
molecules
is occupied by adenine, or a position in each of two polypepddes is occupied
by a lysine),
then the respective molecules are homologous at that position. The percentage
homology
between two sequences is a function of the number of matching or homologous
positions
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shared by the two sequences divided by the number of positions compared x 100.
For
instance, if 6 of 10 of the positions in two sequences are matched or are
homologous, then
the two sequences are 60°~o homologous. By way of example, the DNA
sequences
CTGACT and CAGGTT share 50% homology (3 of the 6 total positions are matched).
Generally, a comparison is made when two sequences are aligned to give maximum
homology. Such alignment can be provided using. for instance, the method of
Needleman
et al., J. Mol Biol. 48: 443-453 (1970), implemented conveniently by computer
programs
such as the Align program (DNAstar, Inc.). Homologous sequences share
identical or
similar amino acid residues, where similar residues are conservative
substitutions for, or
to "allowed point mutations" oG corresponding amino acid residues in an
aligned reference
sequence. In this regard, a "conservative substitution" of a residue in a
reference sequence
are those substitutions that are physically or functionally similar to the
corresponding
reference residues, e.g., that have a similar size, shape, electric charge,
chemical properties,
including the ability to form covalent or hydrogen bonds, or the like.
Particularly preferred
conservative substitutions are those fulfilling the criteria defined for an
"accepted point
mutation" in Dayhoff et al., 5: Atlas of Protein Sequence and Structure, 5:
Suppl. 3,
chapter 22: 354-352, Nat. Biomed. Res. Foundation, Washington, D.C. (1978).
"Homology" and "identity" each refer to sequence similarity between two
polypeptide
sequences, with identity being a more strict comparison. Homology and identity
can each
2o be determined by comparing a position in each sequence which may be aligned
for
purposes of comparison. When a position in the compared sequence is occupied
by the
same amino acid residue, then the poiypeptides can be referred to as identical
at that
position; when the equivalent site is occupied by the same amino acid (e.g.,
identical) or a
similar amino acid (e.g., similar in steric and/or electronic nature), then
the molecules can
be refered to as homologous at that position. A percentage of homology or
identity
between sequences is a function of the number of matching or homologous
positions
shared by the sequences. An "unrelated" or "non-homologous" sequence shares
less than
40 percent identity, though preferably less than 25 percent identity, with an
AR sequence of
the present invention.
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Various alignment algorithms and/or programs may be used, including FASTA,
BLAST or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence
analysis package (University of Wisconsin, Madison, Wis.), and can be used
with, e.g.,
default settings. ENTREZ is available through the National Center for
Biotechnology
Information, National Library of Medicine, National Institutes of Health,
Bethesda, Md. In
one embodiment, the percent identity of two sequences can be determined by the
GCG
program with a gap weight of 1, e.g., each amino acid gap is weighted as if it
were a single
amino acid or nucleotide mismatch between the two sequences.
The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used
interchangeably
o herein. The terms "polynucleotide sequence" and "nucleotide sequence" are
also used
interchangeably herein.
A molecule of the invention is in an "effective amount" if it is that amount
which
produces a result or exerts an influence on the particular condition being
treated.
A "polymer" is a Larger molecule constructed from many smaller structural
units
t 5 called "monomers", linked together in any conceivable pattern. When only
one species of
monomer is used to build a larger molecule, the product is called a
"homopolymet', used
interchangeably with "polymer". If the chains are composed of more than one
different
monomer, the material is generically called a "heteropolymer". The polymer
moiety to
which is attached a CD2 binding agent or fragment or variant is preferably a
polyalkylene
2o glycol polymer but any polymer backbone can be used, most preferably those
that are water
soluble, non-toxic, and non-immunogenic.
Practice of the present invention will employ, unless indicated otherwise,
conventional techniques of cell biology, cell culture, molecular biology,
microbiology,
recombinant DNA, protein chemistry, and immunology, which are within the skill
of the
25 art. Such techniques are described in the literature. See, for example,
Molecular Cloning:
A Laboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.), Cold
Spring
Harbor Laboratory Press,1989; DNA Cloning, Volumes I and II (D.N. Glover, ed),
1985;
Oligonucleotide Synthesis, (M.J. Gait, ed.),1984; U.S. Patent No. 4,683,195
(Mullis et
al.,); Nucleic Acid Hybridization (B.D. Hames and S.J. Higgins, eds.),1984;
30 Transcription and Translation (B.D. Hames and S.J. Higgins, eds.),1984;
Culture of
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Animal Cells (R.I. Freshney, ed). Alan R. Liss, Inc.,1987: Immobilized Cells
and
Enzymes, IRL Press,1986; A Practical Guide to Molecular Cloning (B.
Perbal),1984;
Methods in Enzymology, Volumes 154 and 155 (Wu et al., eds), Academic Press,
New
York: Gene Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos,
eds.),
1987, Cold Spring Harbor Laboratory; Immunochemical Methods in Cell and
Molecular
Biology (Mayer and Walker, eds.), Academic Press, London,1987; Handbook of
Experiment Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds.),
1986;
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press,1986.
While not wishing to be bound by theory, applicants believe that CD2 binding
l0 agents used in accordance with the methods of this invention are
prophylactic and
therapeutic for the treatment of the aforementioned conditions because they
either
modulate the LFA3/CD2 interaction and/or modulate CD2 signaling, resulting in,
among
other things. an inhibition of T cell proliferation and activation and, more
particularly, a
modulation in the number and/or distribution of memory effector T lymphocytes.
Applicants believe that adverse effects of conditions of the type discussed
herein
are due to such T cell proliferation and activation. Applicants believe that
the methods of
the present invention are superior to previously available therapies for these
conditions for
a number of reasons, including, inhibition of antigen specific interactions
for all antigens
present, inhibition of T cell activation, no general immunosuppression and,
possibly,
induction of tolerance. In particular, applicants believe that use of the
methods of this
invention will result in more specific targeting of therapy to T cells
actually in the initiating
stage of the lesion with no effect on polymorphonuclear leukocytes or
macrophage
mediated effector mechanisms. Accordingly, the patient will be less
susceptible to
infections than with steroids or other general immunosuppressants. Thus,
methods of
inhibiting T cell activation, as provided herein, are prophylactic and
therapeutic for such
skin conditions.
CD2 Binding Agents
CD2 binding agents useful in the methods of the invention have their salient
property, the ability to modulate CD2 signaling and to modulate, and
preferably inhibit the
CD2/LFA-3 interaction. A given CD2 binding agent may have the ability to
perform both
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these functions. Sueh agents include anti-LFA-3 antibody homologs, anti-CD2
antibody
homologs, soluble LFA-3 polypeptides, soluble CD2 polypeptides, LFA-3 and CD2
mimetic agents and derivatives thereof. Preferred CD2 binding agents are
soluble CD2 and
LFA-3 polypeptides, anti-CD2 antibody homologs and LFA-3 and CD2 mimetic
agents.
The utility in the methods of this invention of specific soluble CD2
polypeptides,
soluble LFA-3 polypeptides, anti-LFA-3 antibody homologs, anti-CD2 antibody
homologs
or CD2 and LFA-3 mimetic agents rnay be determined, for example, by assaying
their
ability to modulate the LFA-3/CD2 interaction. This ability may be assayed,
for example,
using a simple cell binding assay that permits visual (under magnification)
evaluation of
to the ability of the putative CD2 binding agent to inhibit the interaction
between LFA-3 and
CD2 on cells bearing these molecules. Jurkat cells are preferred as the CD2+
substrate and
sheep red blood cells or human JY cells are preferred as the LFA-3+ substrate.
The
binding characteristics of soluble polypeptides, antibody homologs and mimetic
agents
useful in this invention may be assayed in several known ways, such as by
radiolabeling the
antibody homolog, polypeptide or agent (e.g., 35S or 1251) and then contacting
the labeled
polypeptide, mimetic agent or antibody homolog with CD2+ of LFA-3+ cells, as
appropriate. Binding characteristics may also be assayed using an appropriate
enzymatically labelled secondary antibody. Rosetting competition assays such
as those
described by Seed et al. (ProcyNatl. Acad, Sci. U A, 84, pp. 3365-69 (1987))
may also be
2o used.
A. Anti-LFA-3 And Anti-CD2 Antibody Homologs
Many types of anti-LFA-3 or anti-CD2 antibody homologs are useful in the
methods of this invention. These include monoclonal antibodies, recombinant
antibodies,
chimeric recombinant antibodies, humanized recombinant antibodies, as well as
antigen-
binding portions of the foregoing.
Among the anti-LFA-3 antibody homologs, it is preferable to use monoclonal
anti-
LFA-3 antibodies. It is more preferable to use a monoclonal anti-LFA-3
antibody
produced by a hybridoma selected from the group of hybridomas having Accession
Nos.
ATCC HB 10693 ( 1 E6), ATCC HB 10694 (HC- 1 B 11 ), ATCC HB 10695 (7A6), and
ATCC HB 10696 (8B8), or the monoclonal antibody known as TS2/9 (Sanchez-Madrid
et
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al., "Three Distinct_Antigens Associated with Human T-Lymphocyte-Mediated
Cytolysis:
LFA-1, LFA-2 and LFA-3", Proc. Natl, Acad. Sci, USA, 79, pp. 7489-93 ( 1982)).
Most
preferably, the monoclonal anti-LFA-3 antibody is produced by a hybridoma
selected from
the group of hybridomas having Accession Nos. ATCC HB 10695 (7A6) and ATCC HB
10693 (I E6).
Among the anti-CD? antibody homologs, it is preferable to use monoclonal anti-
CD2 antibodies, such as the anti-CD2 monoclonal antibodies known as the TI 1
epitope ,
antibodies, including TS2/18 (Sanchez-Madrid et al., "Three Distinct Antigens
Associated
with Human T-Lymphocyte-Mediated Cytolysis: LFA- 1, LFA-2 and LFA-3 ", Proc,
Natl.
o Acad. Sci. USA, 79, pp. 7489-93 ( 1982)).
The technology for producing monoclonal antibodies is well known. Briefly, an
immortal cell line (typically myeloma cells) is fused to lymphocytes
(typically splenocytes)
from a mammal immunized with preparation comprising a given antigen, and the
culture
supernatants of the resulting hybridoma cells are screened for antibodies
against the
antigen. See generally, Kohler et al., Nature, "Continuous Cultures of Fused
Cells
Secreting Antibody of Predefined Specificity", 256, pp. 495-97 ( 1975). Useful
immunogens for the purpose of this invention include CD2- or LFA-3-bearing
cells, as
well as cell free preparations containing LFA-3, CD2 or counter receptor-
binding
fragments thereof (e.g., CD2 fragments that bind to LFA-3 or LFA-3 fragments
that bind to
2o CD2).
Immunization may be accomplished using standard procedures. The unit dose and
immunization regimen depend on the species of mammal immunized, its immune
status,
the body weight of the mammal, etc. Typically, the immunized mammals are bled
and the
serum from each blood sample is assayed for particular antibodies using
appropriate
screening assays. For example, useful anti-LFA-3 or anti-CD2 antibodies may be
identified by testing the ability of the immune serum to block sheep red blood
cell resetting
of Jurkat cells, which results from the presence of LFA-3 and CD2 on the
respective
surfaces of these cells. The lymphocytes used in the production of hybridoma
cells
typically are isolated from immunized mammals whose sera have already tested
positive
for the presence of the desired antibodies using such screening assays.
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Typically, the immortal cell line (e.g., a myeloma cell line) is derived from
the
same mammalian species as the lymphocytes. Preferred immortal cell lines are
mouse
myeloma cell lines that are sensitive to culture medium containing
hypoxanthiae,
aminopterin and thymidine ("HAT medium"). Typically, HAT-sensitive mouse
myeloma
s cells are fused to mouse splenocytes using polyethylene Glycol ("PEG") 3350.
Hybridorna
cells resulting from the fusion are then selected using HAT medium, which
kills ~unfused
and unproductively fused myeloma cells (unfiised splenocytes die after several
days
because they are not transformed). Hybridomas producing a desired antibody are
detected
by screening the hybridoma culture supernatants, for example, for the ability
to bind to
their respective counter receptor, or for their ability to block Jackal cell
adhesion to sheep
red blood cells. Subcloning of the hybridoma cultures by limiting dilution is
typically
performed to ensure monoclonality.
To produce anti-LFA-3 or anti-CD2 monoclonal antibodies, hybridnma cells that
tested positive in such screening assays are cultured in.a nutrient medium
under conditions
t5 and for a time sufficient to allow the hybridoma cells to secrete the
monoclonal antibodies
into the culture medium. Tissue culture techniques and culture media suitable
for
hybridoma cells are well known. The conditioned hybridoma culture supernatant
may be
collected and the desired antibodies optionally further purified by well-known
methods.
Alternatively, the desired antibody may be produced by injecting the hybridoma
2o cells into the peritoneal cavity of a pristane-primed mouse. The hybridoma
cells proliferate
in the peritoneal cavity, secreting the antibody, which accumulates as ascites
fluid. The
antibody may be harvested by withdrawing the ascites fluid,from the peritoneal
cavity with
a syringe.
Anti.CD2 and anti-I:.FA-3 antibody homologs useful in the present invention
may
25 also be recombinant aatibodies~produced by host cells transformed with DNA
encoding
immunoglobulia light and heavy.chains of a desired antibody. Recombinant
antibodies
may be produced by well known genetic engineering technic~es. See, e.g., U.S:
Patent No.
4,81b,397:.- 1~or example, recombinant antibod:ie~
may be produced by cloning cDNA or genomic DNA encoding the immunoglobulin
light
3o and heavy chains of the desired antibody from a hybridoma cell that
produces an antibody
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homolog useful in this invention. The cDNA or genomic DNA encoding those
polypeptides is then inserted into expression vectors so that both genes are
operatively
linked to their own transcriptional and translational expression control
sequences. The
expression vector and expression control sequences are chosen to be compatible
with the
expression host cell used. Typically, both genes are inserted into the same
expression
vector. Prokaryotic or eukaryotic host cells may be used. Expression in
eukaryotic host
cells is preferred because such cells are more likely than prokaryotic cells
to assemble and
secrete a properly folded and immunolo~ically active antibody. However, any
antibody
produced that is inactive due to improper folding may be renaturable according
to well
~o known methods (Kim and Baldwin, "Specific Intermediates in the Folding
Reactions of
Small Proteins and the Mechanism of Protein Folding". Ann, Rev. Biochem., S 1,
pp. 459-
89 ( 1982)). It is possible that the host cells will produce portions of
intact antibodies, such
as light chain dimers or heavy chain dimers, which also are antibody homologs
according
to the present invention.
i5 It will be understood that variations on the above procedure are useful in
the present
invention. For example, it may be desired to transform a host cell with DNA
encoding
either the light chain or the heavy chain (but not both) of an antibody
homolog.
Recombinant DNA technology may also be used to remove some or all of the DNA
encoding either or both of the light and heavy chains that is not necessary
for CD2 or LFA-
20 3 counter receptor binding. The molecules expressed from such truncated DNA
molecules
are useful in the methods of this invention. In addition, bifunctionai
antibodies may be
produced in which one heavy and one light chain are anti-CD2 or anti-LFA-3
antibody
homologs and the other heavy and light chain are specific for an antigen other
than CD2 or
LFA-3, or another epitope of CD2 or LFA-3.
25 Chimeric recombinant anti-LFA-3 or anti-CD2 antibody homologs may be
produced by transforming a host cell with a suitable expression vector
comprising DNA
encoding the desired immunoglobulin light and heavy chains in which all or
some of the
DNA encoding the hinge and constant regions of the heavy and/or the light
chain have
been substituted with DNA from the corresponding region of an immunoglobulin
light or
3o heavy chain of a different species. When the original recombinant antibody
is nonhuman,
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and the CD2 binding agent is to be administered to a human, substitution of
corresponding
human sequences is preferred. An exemplary chimeric recombinant antibody has
mouse
variable regions and human hinge and constant regions. See generally, U.S.
Patent No.
4,816.397 and Morrison et al., "Chimeric Human Antibody Molecules: Mouse
Antigen-
s Binding Domains With Human Constant Region Domains", Proc. Natl. Acad. Sci.
USA,
8 1, pp. 6851-55 ( 1984). Humanized recombinant anti-LFA-3 or anti-CD2
antibodies may
be produced by transfotTrting a host cell with a suitable expression vector
comprising DNA
encoding the desired nonhuman immunoglobulin light and heavy chains in which
all or
some of the DNA encoding amino acids not involved in antigen binding have been
t0 substituted with DNA from the corresponding region of a desired human
immunoglobulin
light or heavy chain. See generally, Jones et al., "Replacing the
Complementarity-
Determining Regions in a Human Antibody with Those from a Mouse", Nature, 321,
pp.
522-25 ( 1986).
Anti-CD2 and anti-LFA-3 antibody homologs that are not intact antibodies are
also
I5 useful in this invention. Such homologs may be derived from any of the
antibody
homologs described above. For example, antigen-binding fragments, as well as
full-length
monomeric, dimeric or trimeric polypeptides derived from the above-described
antibodies
are themselves useful. Useful antibody homologs of this type include Fab
fragments,
Fab'fragments, F(ab~2 fragments, F(v) fragments, heavy chain monomers or
dimers, light
2o chain monomers or dimers, dimers consisting of one heavy and one light
chain, and the
like. Anti-LFA-3 heavy chains are preferred anti-LFA-3 antibody fragments.
Antibody fragments may also be produced by chemical methods, e.g., by cleaving
an intact antibody with a protease, such as pepsin or papain, and optionally
treating the
cleaved product with a reducing agent. Alternatively, useful fragments may be
produced
25 by using host cells transformed with truncated heavy and/or light chain
genes. Heavy and
light chain monomers may be produced by treating an intact antibody with a
reducing
agent, such as dithiothreitol, followed by purification to separate the
chains. Heavy and
light chain monomers may also be produced by host cells transformed with DNA
encoding
either the desired heavy chain or light chain, but not both. See, e.g., Ward
et al., "Binding
30 Activities of a Repertoire of Single Immunoglobulin Variable Domains
Secreted from
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.y
Escherichia coli", Nature. 341, pp. 544-46 (1989); Sastry et al., "Cloning of
the
Immunological Repertoire in Escherichia coli for Generation of Monoclonal
Catalytic
Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA
Library",
Proc. Natl. Acad. Sci. USA, 86, pp. 572832 ( 1989).
B. Soluble CD2 and LFA-3 Polypeptides
Soluble LfiA-3 polypeptides or soluble CD2 polypeptides that modulate the
interaction of LFA-3 and CD2 are useful in the methods of the present
invention. Soluble
LFA-3 polypeptides ate preferred.
t0 Soluble LFA-3 polypeptides may be derived from the transmembrane form of
LFA-
3, particularly the extracellular domain (e.g.,AA 1-AA187 of SEQ 1D N0:2).
Such
polypeptides are described in U.S. Patent No. 4,956,281 and U.S. Patent No:
5,547,853
(which shares a common assignee,with the present application).:
Prefe.~red soluble LPA-3 polypeptide include polypelatide.s
consisting of AA i-AA 92 of SEQ >D N0:2, AA 1-AA 80 of SEQ >D N0:2, AA 50-AA
65
of SEQ ID N0:2 and AA 20-AA 80 of SEQ ID N0:2. A vector comprising a DNA
sequence (SEQ m NO: 1 ) that encodes the amino acids of SEQ ID N0:2 has been
deposited with the American Type Culture Collection, Rockville, Maryland under
Accession No. 75107.
2o The most preferred proteins of this type are fusion proteins that contain
the amino
terminal 92 amino acids of mature LFA-3, the C-terminal 10 amino acids of a
human IgG 1
hinge region containing the two cysteine residues thought to participate in
interchain
disulfide bonding, and the CH2 and CH3 regions of a human IgGI heavy chain
constant
domain (e.g., SEQ ID NO: 8). This fusion protein is referred to herein as
°1.FA3TIP." A
plasmid, pSABI52, encoding an exemplary LFA3TIP has been deposited with the
American Type Culture Collection, Rockville, Maryland,~under tlu accession
number
ATCC 68720: The DNA sequence of the pSAB 152 insert is SEQ ID N0:7. This
recombinant protein has been designed to-modulate immune responses through
interaction .
with the CD2 receptor. The LFA-3 portion of the fusion binds to the CD2
receptor on T
lymphocytes. The IgGi portion binds to the FcgammaRI (macrophage) and
FcgammaRlll
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(NK cells and neuttophils) receptors. The interaction of LFA3TIP with CD2 has
been
shown to inhibit in vitro human T lymphocyte responses. See United States
Patent
5,728,677 and 5,547,853. One way of producing LFA3TIP for use in the methods
of this
invention is described in United States Patent 5,547,853.
Generally, conditioned culture medium of COS7 or CHO cells transfected with
pSAB 152 is concentrated using an AMICON SI Y3 0 spiral cartridge system
(AMICON,
Danvers, Massachusetts) and subjected to Protein A-Sepharose 4B (Sigma, St.
Louis,
Missouri) chromatography. The bound proteins are eluted and subjected to
Superose-12
(Pharmacia/LKB, Piscataway, New Jersey) gel filtration chromatography.
Superose-12
tU fractions containing LFA3TIP with the least amount of contaminating
proteins, as
determined on SDS-PAGE gels and by Western blot analysis, (see, e.g., Towbin
et al.,
Proc. Natl. Acad. Sci. USA, 74, pp. 4350-54 (1979); Antibodies: A Laboratory
Manual,
pp. 474-5 1 0 (Cold Spring Harbor Laboratory (I 98 8)), are pooled and
concentrated in a
YM30 Centricon (AMICON). LFA3TIP is detected on Western blots using a rabbit
anti-
~5 LFA-3 polyclonal antiserum, followed by delectably labeled goat anti-rabbit
IgG. The
purified LFA3TIP of COS7 or CHO cells is a dimer of two monomeric LFA-3-Ig
fusion
proteins, connected by disulfide bonds.
Another preferred fusion protein consists of the first and second LFA-3
extracellular domains fused to the hinge CH2 and CH3 regions of human IgGI,
herein
2o referred to as LLFA3-Ig.
Soluble LFA-3 polypeptides may also be derived from the PI-linked form of LFA-
3, such as those described in PCT Patent Application Serial No. WO 90/0218 1.
A vector
comprising a DNA sequence encoding PI-linked LFA-3 (i.e., SEQ lD N0:3) is
deposited
with the American Type Culture Collection, Rockville, Maryland under Accession
No.
25 68788. It is to be understood that the PI-linked form of LFA-3 and the
transmembrane
form of LFA-3 have identical amino acid sequences through the entire
extracellular
domain.
Accordingly, the preferred PI-linked LFA-3 polypeptides are the same as for
the
transmembrane form of LFA-3. Soluble CD2 polypeptides may be derived from full
length
3o CD2, particularly the extracellulardomain (e.g.,AA I-AA 185 of SEQ ll~
N0:6). Such
CA 02514899 1999-08-31
polypeptides may comprise all or part of the exuacellular domain of CD2.
Exemplary
soluble CD2 polypeptides are described in PCT WO 90/08187.
The production of the soluble polypeptides.useful in this invention may be
achieved
by a variety of methods known in the art. For example; the polypeptides may be
derived
from intact transmembrane LFA-3 or CD2 molecules or an intact PI-linked LFA~3
molecule by proteolysis using specific endopeptidases in combination with
exopeptidases,
Bdman degradation, or both. The intact LFA-3 molecute or the intact CD2
molecule, in
turn, inay be purified from its natural source using conventional methods. .
Alternatively.
!o the intact LFA-3 or CD2 may be produced by known recombinant DNA techniques
using
cDNAs (see; e.g., U.S. Patent No. 4,956,281 to Wallner et al.; Aruffo and
Seed, Proc. Natl.
Acad. Sci.,.84, pp. 2941-4.5 (1987); Sayre et al., Proc. Natl. Acad. Sci. USA,
84, pp.
2941-45 ( 1987)).
Preferably, the soluble polypeptides useful in the present invention are
produced
l5 directly, thus eliminating the need for an entire LFA-3 molecule or an
entire CD2 molecule
as a starting material. This may be achieved by conventional chemical
synthesis
techniques or by well-_k_nown _recembinaut DNA !echniques wherein only these
DNA
sequences which encode the desired peptides are expressed in transformed
hosts. For
example, a gene which encodes the desired soluble LFA-3 polypeptide or soluble
CD2
2o polypeptide may be synthesized by chemical means using an oligonucleotide
synthesizer.
Sucholigonucleotides are designed based on the amino acid sequence of the
desired
soluble LFA-3 polypeptide or soluble CD2 polypeptide. Specific DNA sequences
coding
for the desired peptide also can be derived from the-full length DNA sequence
by isolation
of specific restriction endonuclease fragments or by PCR synthesis of the
specified region.
25 Standard methods maybe applied to synthesize a gene encoding a soluble LFA-
3
polypeptide or a soluble CD2 polypeptide that is useful ici this invention.
For example, the
complete amino acid sequence may 15e used to construct a b~ek-translated gene.
A DNA
oligomer containing a nucleotide sequence coding for a soluble LFA-3
polypeptide or a
soluble CD2 polypeptide useful in-this invention may be synthesized in a
single step.
30 Alternatively, several smaller oligonucleotides coding 'for portions of
the'desired
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polypeptide may be synthesized and then ligated. Preferably, a soluble LFA-3
polypeptide
or a soluble CD2 polypeptidc useful in this invention will be synthesized as
several
separate oligonucleotides which are subsequently linked together, The
individual
oligonucleotides typically contain 5' or 3' overhangs for complementary
assembly. Once
assembled, preferred genes will be characterized by sequences that are
recognized by
restriction endonucleases (including unique restriction sites for direct
assembly into a
cloning or an expression vector), preferred codons taking into consideration
the host
expression system to be used, and a sequence which, when transcribed, produces
a stable,
efficiently translated MRNA. Proper assembly may be confirmed by nucleotide
sequencing, restriction mapping, and expression of a biologically active
polypeptide in a
suitable host.
It will be appreciated by those of skill in the art that, due to the
degeneracy of the
genetic code, DNA molecules comprising many other nucleotide sequences will
also be
capable of encoding the soluble LFA-3 and CD2 polypeptides encoded by the
specific
DNA sequences described above. These degenerate sequences also code for
polypeptides
that are useful in this invention.
The DNA sequences may be expressed in unicellular hosts. As is well known in
the art, in order to obtain high expression levels of a transfected gene in a
host, the gene
must be operatively linked to transcriptional-and translational expression
control sequences
2o that are functional in the chosen expression host. Preferably, the
expression control
sequences, and the gene of interest, will be contained in an expression vector
that further
comprises a bacterial selection marker and origin of replication. If the
expression host is a
eukaryotie cell, the expression vector should further comprise an additional
expression
marker useful in the expression host. The DNA sequences encoding the desired
soluble
polypeptides may or may not encode a signal sequence. If the expression host
is
prokaryotic, it generally is preferred that the DNA sequence not encode a
signal sequence.
If the expression host is eukaryotic, it generally is preferred that a signal
sequence be
encoded, An amino terminal methionine may or may not be present on the
expressed
product. If the terminal methionine is not cleaved by the expression host, it
may, if
desired, be chemically removed by standard techniques.
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A wide variety of expression host/vector combinations may be employed. Useful
expression vectors for eukaryotic hosts, include, for example, vectors
comprising
expression control sequences from SV40, bovine papilloma virus, adenovirus and
cytomegalovirus. Useful expression vectors for bacterial hosts include known
bacterial
plasmids, such as plasmids from E. coli, including col EI, PCRI, pBR322, pMB9
and their
derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the
numerous
derivatives of phage lambda, e. g., NM989, and other DNA phages, such as M13
and
filamentous single stranded DNA phages. Useful expression vectors for yeast
cells include
the ?u plasmid and derivatives thereof. Useful vectors for insect cells
include pVL 941. In
l0 addition, any of a wide variety of expression control sequences may be used
in these
vectors. Such useful expression control sequences include the expression
control
sequences associated with structural genes of the foregoing expression
vectors. Examples
of useful expression control sequences include, for example, the early and
late promoters of
SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the
major
operator and promoter regions of phage lambda, the control regions of fd coat
protein, the
promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid
phosphatase, e.g., PhoS, the promoters of the yeast a-mating system and other
sequences
known to control the expression of genes of prokaryotic or eukaryotic cells or
their viruses,
and various combinations thereof.
2o A wide variety of unicellular host cells are useful. These hosts may
include well
known eukaryotic and prokaryotic hosts, such as strains of E. coli,
PseudomonasLBacillus,
Streptomvces, fungi, yeast, insect cells such as ~odoEtera fru~inerda (SF9),
animal cells
such as CHO and mouse cells, African green monkey cells such as COS l, COS 7,
BSC 1,
BSC 40, and BMT 10, and human cells, as well as plant cells in tissue culture.
For animal
cell expression, we prefer C 140 cells and COS 7 cells. It should, of course,
be understood
that not all vectors and expression control sequences will function equally
well to express
the DNA sequences described herein. Neither will all hosts function equally
well with the
same expression system. However, one of skill in the art may make a selection
among
these vectors, expression control sequences and hosts without u~idue
experimentation. For
3o example, in selecting a vector, the host must be considered because the
vector must
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-'_'S-
replicate in it. The-vector's copy number, the ability to control that copy
number, and the
expression of any other proteins encoded by the vector, such as antibiotic
markers, should
also be considered.
In selecting an expression control sequence, a variety of factors should also
be
considered. These include, for example, the relative strength of the sequence,
its
controllability, and its compatibility with the DNA sequences discussed
herein, particularly
as regards potential secondary structures. Unicellular hosts should be
selected by
consideration of their compatibility with the chosen vector, the toxicity of
the product
coded for by the DNA sequences, their secretion characteristics, their ability
to fold the
soluble polypeptides correctly, their fermentation or culture requirements,
and the ease of
purification of the products coded for by the DNA sequences. Within these
parameters, one
of skill in the art may select various vectorlexpression control sequence/host
combinations
that will express the desired DNA sequences on fermentation or in large scale
animal
culture, for example with CHO cells or COS 7 cells.
The soluble LFA-3 and CD2 polypeptides may be isolated from the fermentation
or
cell culture and purified using any of a variety of conventional methods. One
of skill in the
art may select the most appropriate isolation and purification techniques.
While recombinant DNA techniques are the preferred method of producing useful
soluble CD2 polypeptides or soluble LFA-3 polypeptides having a sequence of
more than
20 amino acids, shorter CD2 or LFA-3 polypeptides having less than about 20
amino acids
are preferably produced by conventional chemical synthesis techniques.
Synthetically
produced polypeptides useful in this invention can advantageously be produced
in
extremely high yields and can be easily purified. Preferably, such soluble CDZ
polypeptides or soluble LFA-3 polypeptides are synthesized by solution phase
or solid
phase polypeptide synthesis and, optionally, digested with carboxypeptidase
(to remove C-
terminal amino acids) or degraded by manual Edman degradation (to remove N-
terminal
amino acids). Proper folding of the polypeptides may be achieved under
oxidative
conditions which favor disulfide bridge formation as described by Kent,
"Chemical
Synthesis of Polypeptides and Proteins", Ann. Rev. Biochem., 57, pp. 95789
(1988).
Polypeptides produced in this way may then be purified by separation
techniques widely
CA 02514899 1999-08-31
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known in the art, preferably utilizing reverse phase HPLC. The use of solution
phase
synthesis advantageously allows for the direct addition of certain
.derivatized amino acids
to the growing polypeptide chain, such as the 0-sulfate ester of tyrosine.
This obviates the
need for a subsequent derivatization step to modify.any residue of the
polypeptides useful
5 in this invention.
C. LFA-3 And CD2 Mimetic A,~n~s
Also useful in the methods of this invention are LFA-3 and CD2 mimetic agents.
These agents which may be peptides, semi-peptidic compounds or non-peptidie
t0 compounds, are CD2 biding agents that modulate CD2 signaling and/or the
CD2/LFA-3
interaction. The most preferred CD2 and LFA-3 mimetic agents will inhibit the
CD2/LFA-
3 interaction at least as well as anti-LFA-3 monoclonal antibody 7A6 or anti-
CD?
monoclonal antibody TS2/18 (described supra). Such mimetic agents may be
produced by
synthesizing a plurality of peptides (e.g., 520 amino acids in length); semi-
peptidic
15 compounds, or non-peptidic; organic compounds, and then screening those
compounds for
their ability to inhibit the CD2/L,FA-3 interaction. See generally U.S..Patent
No. 4,833,092,
Scott and Smith, "Searching. for Peotide I:igands .with an Epitope ~.ibrary",
Ss.~'en~, 249,
pp. 386-90 ( 1990), and Devlin et al., "Random Peptide Libraries: A-Source of
Specific
Protein Binding Molecules", Science, 249, pp. 40407 ( 1990) .
20
D. Derivatized CD2 binding_agents
Also useful in the methods of this invention are derivatized CD2 binding
agents
such as modulators of the CD2/LFA-3 interaction in which, for example, any of
the
25 antibody homologs, soluble CD2 and~LFA-3 polypeptides,.or CD2.and LFA-3
mimetic
agents described herein are functionally linked (by chemical coupling, genetic
fusion or
otherwise) to one or more members'independently selected'~om the group
consisting of
anti-LFA-3 and anti-CD2 antibody homol~ogs, soluble LFA-3 and CD2
polypeptides, CD2
and LFA-3 mimetic agents, cytotoxic agents and pharmaceutical agents. One type
of
3o derivatized CD2 binding agent is produced by crosslinking two or more CD2
binding
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agents (of the same type or of different types). Suitable crosslinkers include
those that are
heterobif mctional, having two distinctly reactive groups separated by an
appropriate
spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or
homobiftmctional (e.g.,
disuccinimidyl suberate). Such linkers are available from Pierce Chemical
Company,
Rockford, lllinois: Another possibility for cross-linking takes advantage of
the PI linkage
signal sequence in PI-linked LFA-3, or fragments thereof. Specifically, DNA
encoding the
PIlinkage signal sequence (e.g., AA162-AA212 of SEQ iD N0:4) is ligated
downstream of
DNA encoding a desired polypeptide, preferably a soluble LFA-3 polypeptide. If
this
construct is expressed in an appropriate eukaryotic cell, the cell will
recognize the PI
t0 linkage signal sequence and will covalently link PI to the polypeptide. The
hydrophobic
property of the PI may then be exploited to form micellar aggregates of the
polypeptides.
Also useful are CD2 binding agents linked to one or more cytotoxic or
pharmaceutical agents. Useful pharmaceutical agents include biologically
active peptides,
polypeptides and proteins, such as antibody homologs specific for a human
polypeptide
t5 other than CD2 or LFA-3, or portions thereof Useful pharmaceutical agents
and cytotoxic
agents also include cyclosporin A, prednisone, FK506, methotrexate, steroids,
retinoids,
interferon, and nitrogen mustard.
Preferred CD2 binding agents derivatized with a pharmaceutical agent include
recombinantly produced polypeptides in which a soluble LFA-3 polypeptide,
soluble CD2
20 polypeptide, or a peptidyl CD2 or peptidyl LFA-3 mimetic agent is fused to
all or part of an
immunoglobulin heavy chain hinge region and all or part of a heavy chain
constant region.
Preferred polypeptides for preparing such fusion proteins are soluble LFA-3
polypeptides.
Most preferred are fusion proteins containing AA 1-AA 92 of LFA-3 (e.g., SEQ m
N0:2)
fused to a portion of a human IgG I hinge region (including the C-terminal ten
amino acids
25 of the hinge region containing two cysteine residues thought to participate
in interehain
disulf de bonding) and the CH2 and CH3 regions of an IgGI heavy chain constant
domain.
Such fusion proteins are expected to exhibit prolonged serum half lives and
enable CD2
binding agent dimerization.
Other derivatized CD2 binding agents also expected to exhibit prolonged serum
30 half lives are CD2 binding agents linked most preferably to one or more
polymers
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comprising a poIyalkylene glycol polymer. The polymer would be expected to
selectively
react with free amino or other reactive groups on a CD2 binding agent and in
theory, the
polymers) are reacted so that attachment could occur at any available amino
group such as
alpha amino groups or the epsilon-amino groups of lysines, or -SH groups of
cysteines.
Free carboxylic groups, suitably activated carbonyl groups, hydroxyl,
guanidyl, oxidized
carbohydrate moieties and mercapto groups of the CD2 binding agent (if
available) can
also be used as attachment sites.
Irt particular, if the CD2 binding agent is a protein, the chemical
modification of
any cysteine (e.g., an internal or an N-terminal cysteine) to protect the
thiol, with
to concomitant conjugation with a polyalkylene glycol moiety (i.e.,
polyethylene glycol or
PEG), can be carried out in numerous ways by someone skilled in the art. See
United
States Patent 4,179,337. The sulfhydryl moiety, with the thiolate ion as the
active species,
is the most reactive functional group in a protein. There are many reagents
that react faster
with the thiol than any other groups. See Chemistry of Protein Conjugation and
Cross-
~5 Linking (S. S. Wong, CRC Press, Boca Raton, FL, 1991).
Other examples of methods that provide linkage between a polyalkylene glycol
and a
cysteine would be reactions with other alpha-haloacetyl compounds,
organomercurials,
disulfide reagents, and other N-substituted maleimides. Numerous derivatives
of these
active species are available commercially (e.g., ethyl iodoacetate (Aldrich,
Milwaukee WI),
20 phenyl disulfide (AIdrich), and N-pyrenemaleimide (Molecular Probes, Eugene
OR)) or
could be synthesized readily (e.g., N-alkyliodoacetamides, N-alkylmaleimides,
and
_ organomercurials). While there is no simple chemical strategy for targeting
a polyalkylene
glycol polymer such as PEG to a CD2 binding agent, it is straightforward to
genetically
engineer a site that can be used to target the polymer moiety, such as using
site-directed
25 mutagenesis. For example, incorporation of a Cys at a site that is at or
near the C-terminus
of a proteinaceous CD2 binding agent allows specific modification using a
maleimide,
vinylsulfone or haloacetate- activated polyalkylene glycol (e.g., PEG). The
reactions may
take place by any suitable method used for reacting biologically active
materials with inert
polymers.
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Generally the process involves preparing an activated polymer (that may have
at least
one terminal hydroxyl group) and thereafter reacting the protein with the
activated polymer
to produce the soluble protein suitable for formulation. The above
modification reaction
can be performed by several methods, which may involve one or more steps. In
the
practice of the present invention, polyalkylene glycol residues of CI-C4 alkyl
polyalkylene
glycols, preferably polyethylene glycol (PEG), or poly(oxy)alkylene glycol
residues of such
glycols are advantageously incorporated in the polymer systems of interest.
Thus, the
polymer to which the protein is attached can be a homopolymer of polyethylene
glycol
(PEG) or is a polyoxyethylated polyol, provided in all cases that the polymer
is soluble in
water at room temperature. Non-limiting examples of such polymers include
polyalkylene
oxide homopolymers such as PEG or polypropylene glycols, polyoxyethylenated
glycols,
copolymers thereof and block copolymers thereof, provided that the water
solubility of the
block copolymer is maintained.
Examples of polyoxyethylated polyols include, for_example, polyoxyethylated
glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or the like.
The glycerol
backbone of polyoxyethylated glycerol is the same backbone occurring naturally
in, for
example, animals and humans in mono-, di-, and triglycerides. Therefore, this
branching
would not necessarily be seen as a foreign agent in the body.
As an alternative to polyalkylene oxides, dextran, polyvinyl pyrrolidones,
2o polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the
like may be
used. Moreover, heteropolymers (i.e., polymers consisting of more than one
species of
monomer such as a copolymer) as described in U.S. Patent 5,359,030 may be used
(e.g.,
proteins conjugated to polymers comprising a polyalkylene glycol moiety and
one or more
fatty acids) Those of ordinary skill in the art will recognize that the
foregoing list is merely
illustrative and that all polymer materials having the qualities described
herein are
contemplated.
Moreover, in another aspect of the invention, one can utilize a derivatized
CD2
binding agent covalently bonded to the polymer component in which the nature
of the
conjugation involves cleavable covalent chemical bonds. This allows for
control in terms
of the time course over which the polymer may be cleaved from the CD2 binding
agent.
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This covalent bond-may be cleaved by chemical or enzymatic reaction. The
polymer-CD2
binding agent product retains an acceptable amount of activity. Concurrently,
portions of
polyethylene glycol are present in the conjugating polymer to endow the
polymer-CD2
binding agent conjugate with high aqueous solubility and prolonged blood
circulation
capability. As a result of these improved characteristics the invention
contemplates
parenteral, aerosol, and oral delivery of both the active polymer-CD2 binding
agent species
and, following hydrolytic cleavage, bioavailability of the,CD2 binding agent
per se, in in
vivo applications.
t0 Pharmaceutical Compositions And Methods According To This Invention
This invention provides a preferred method for preventing or treating a
condition in
a mammal characterized by: the infiltration into one or more organs of the
mammal of
memory effector T lymphocytes; and/or the association with such condition by
such T
lymphocytes- The method includes administering to the mammal one or more CD2
t5 binding agents such as inhibitors of the CD2/LFA-3 interaction, or
derivatized forms)
thereof that are capable of selectively modulating memory effector T
lymphocytes as
compared to naive T lymphocytes. Preferably, an effective amount of the CD2
binding
agent or derivatized form thereof is administered. By "effective amount" is
meant an
amount capable of lessening the spread or severity of the conditions described
herein.
2o It will be apparent to those of skill in the art that the effective amount
of CD2
binding agent will depend, inter alia, upon the administration schedule, the
unit dose
administered, whether the CD2 binding agent is administered in combination
with other
therapeutic agents, the immune status and health of the patient, the
therapeutic activity of
the particular CD2 binding agent administered and the serum half-life.
25 The CD2 binding agent, or pharmaceutical composition, may be in a variety
of
forms. These include, for example, solid, semi-solid and liquid dosage forms,
such as
tablets, pills, powders, liquid solutions, dispersions or suspensions,
liposomes,
suppositories, injectable infusible, and topical preparations. The preferred
form depends
on the intended mode of administration and therapeutic application. The
preferred forms
30 are injectable or infusible solutions. The CD2 binding agent or
pharmaceutical
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composition may be_administered intravenously, intramuscularly,
subcutaneously> intra-
articularly, intrathecally, periostally, intratumorally, intralesionally,
perilesionally by
infusion, orally, topically or by inhalation. Preferably it is administered
subcutaneously,
intramuscularly or intravenously. Most preferably, it is administered
subcutaneously. The
CD2 binding agents may be administered orally, buccally, parenterally,
rectally, vaginally,
by intranasal inhalation spray, by intrapulmonary inhalation or in other ways.
In particular,
the agents according to the invention may be formulated for inhalation with
spray or
powder, for injection (for example subcutaneous, intramuscular, intravenous,
infra-articular
or intracisternal injection), for infusion or for oral administration and may
be presented in
o unit dose form in ampoules or tablets or in multi-dose vials or other
containers with an
added preservative. The compositions may take such forms as suspensions,
solutions, or
emulsions or gels in oily or aqueous vehicles, and may contain formulatory
agents such as
suspending, stabilising and/or dispersing agents. Alternatively, the active
ingredient may be
in powder and/or lyophilised form for direct administration or for
constitution with a
t5 suitable vehicle (e.g. sterile, pyrogen-free water, normal saline or 5%
dextrose) before use.
Preferably, the unit dose is administered about one to three times per week or
one to
three times per day. More preferably, it is administered about one to three
times per day
for between about 3 and 7 days, or about one to three times per day for
between about 3
and 7 days on a monthly basis. It will be recognized, however, that lower or
higher
20 dosages and other administrations schedules may be employed. Preferably,
the CD2
binding agent is administered at a dose between about 0.001 and about 50 mg
CD2 binding
agent per kg body weight, more preferably, between about 0.01 and about 10 mg
CD2
binding agent per kg body weight, most preferably between about 0.1 and about
4 mg CD~
binding agent per kg body weight.
25 For example, to administer a CD2 binding agent of the invention (the
soluble LFA-
3 polypeptide LFA3T1P) to a patient, a particularly preferred CD2 binding
agent is
packaged as a frozen solution in vials containing 10 mg/mL recombinant LFA-
3/IgG,
fusion protein and excipient materials (sodium chloride, monobasic potassium
phosphate,
and dibasic sodium phosphate). The vials are thawed in the refrigerator or at
room
3o temperature and diluted with 0.9% sodium chloride to a final volume of 5 mL
for N bolus
CA 02514899 1999-08-31
administration. LFA3TIP, is constructed as described in Miller et al. ( 1993)
J Fxp. iYled. I
78:211, and is purified from culture medium of
transfectant CHO (Chinese Hamster Ovary) cell lines by absorption to Protein-A
Sepharose
4B (Pharmacia).and eluted with SO mM glycine, 250 mM NaCI (pH 3.0). Fractions
5 containing protein are pooled and subjected to gel filtration on Superose-6~
(Pharmacia) in
phosphate buffered saline (PBS). Peak fractions are pooled and analyzed for
purity on
12°~ reducing and non-reducing SDS-PAGE.
In one exemplary protocol, the patient receive one N bolus every week at a
dosage
_ that preferably ranges greater than 0.025 mg/lg and can be up to.betwecn .
0.075 mg/kg,
1o through 0.150 mg/ha . Dosing of this CD2 binding agent 'is 'based on the
subject's baseline
body weight. Other dosage regimens can include one intramuscular (1M)
injection at a
dosage of no less than 0.005 mg/kg, with preferred dosages ranging upwards of
0.025
mg/I:g, the most preferred dosages being selected from 0.05, 0.075, 0.1125,
and
0.165 mglkg.
15 Unit doses should be administered until an effect is observed. The effect
may be
measured by a variety of methods. ~n the case of MS, the therapeutic effect
can be
rrzeastered by sca~da~d r~,agn~tic resonance tnet_hods. L~ the case of >gD,
assaying extent of
abdominal pain, healing of fistulas, frequency of stools, and the Iike, in the
case of psoriatic
arthritis, by changes-in joint swelling and range in motion. Persons having
ordinary skill in
2o the medical arts will readily be able to measure the clinical effect on any
one of the
indications described herein by reference to well known medical procedures.
The CD2 binding agents) or derivatized forms) thereof-are preferably
.. administered in a composition including a pharmaceutically acceptable
carrier. By
"pharmaceutically acceptable.carrier" is meant a carrier that does not eanse
an allergic
25 reaction or other untoward effect in patients to whom it is administered.
Suitable
pharmaceutically acceptable carriers include, for example; one or more of
water, saline,
phosphate buffered saline, dextrose; glycerol, ethanol and~tl5e like; as well
as combinations
thereof. Pharmaceutically acceptable carriers may fut~ther comprise minor
amounts of
auxiliary substances such as wetting or emulsifying agents, preservatives or
buffers, which
3o enhance the shelf life or effectiveness of the CD2 binding agent.
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The pharmaceutical composition or CD2 binding agent may be administered in
conjunction with other therapeutic or prophylactic agents. These include, for
example,
cyclosporin A, steroids, retinoids, nitrogen mustard, interferon,
methotrexate, antibiotics
and antihistamines. These agents may be administered in single dosage form
with the CD?
binding agent (i.e., as pan of the same pharmaceutical composition), a
multiple dosage
form separately from the CD2 binding agent, but concurrently, or a multiple
dosage form
wherein the two components are administered separately but sequentially.
Alternatively,
the CD2 binding agent and the other active agent may be in the form of a
single conjugated
molecule. Conjugation of the two components may be achieved by standard cross-
linking
to techniques well known in the art. A single molecule may also take the form
of a
recombinant fusion protein. In addition, the CD? binding agents, or
pharmaceutical
compositions, useful in the present invention may be used in combination with
other
therapies such as chemotherapy. Such combination therapies may advantageously
utilize
lower dosages of the therapeutic agents.
Gene Therapy
The invention is based further on gene therapy based delivery of the CD2
binding
agents as expression products of a nucleic acid sequence within a mammalian
host.
Preferred ex vivo systemic delivery of the gene products entails use of a
recombinant viral
or non-viral vector followed by in vitro transfection of specific mammalian
cell
populations, recovery and purification of the transfected cells and
administration to the
patient. In vivo gene therapy entails the same use of vector and transfection
without
removing any cells or tissue from the patient. The specific cell populations
utilized as
targets for transfection by the recombinant vector containing a nucleic acid
sequence of
interest may include, but are not limited to, ( 1 ) bone marrow cell
populations containing
hematopoietic progenitor cells; (2) peripheral blood leukocyte populations,
preferably
CD34 + blood leukocyte populations, which are enriched for hematopoietic cells
and can
be utilized to repopulate the transfected hematopoietic cells upon
introduction into the
patient without ablation; (3) peripheral blood lymphocyte populations; (4)
myoblast cells,
3o which may be transplanted back into the host subsequent to in vitro
transfection of the
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_3a_
nucleic sequence of interest; and (5) delivery of the recombinant viral or non-
viral vector
containing the nucleic acid sequence of interest, or the nucleic acid sequence
itself, by
intramuscular injection.
For example, the ex vivo use of bone marrow containing hematopoietic
progenitor
cells containing a vector capable of encoding upon expression, a CD2 binding
agent is
well within the level of skill in the art. Briefly, bone marrow cells are
removed, infected
with the recombinant virus, and reintroduced back into the mammalian host.
Thus, a gene
or gene fragment is systemically distributed within the mammalian host,
expression of this
DNA sequence resulting in systemic delivery of the gene product within the
mammalian
t o host.
Peripheral blood is a source of mammalian cells for infection by viral
mediated
vectors. More specifically, blood leukocytes, especially the CD34 + population
which
contain the circulating hematopoietic stem cells, may be used as target cells
for viral
infection, followed by repopulation of the mammalian host marrow without
ablation
t5 (Karlsson, et al., 1993. Bone Marrow Transplant. 11(supp. 1): 124-127).
Additionally,
lymphocytes may also be utilized as target cells to promote systemic delivery
of the nucleic
acid sequence of interest. The lymphocytes may be removed from the peripheral
blood of
the host, cultured by known techniques and used as the target cell population
for infection
by viral vectors containing the nucleic acid sequence of interest. The
infected lymphocytes
2o may then be injected into the mammalian host (e.g., see Anderson, et al.,
1990, Human
Gene Therany 1: 331-361). An additional target cell population are myoblasts.
Blood
flow into the relatively large mass of skeletal muscle renders this tissue a
localized
repository for the nucleic acid sequence of interest. Therefore, in vitro
infection and
reintroduction of myoblast cells into the mammalian host provides a local
target within the
25 host which results in systemic delivery of the CD2 product. Myoblast
culture, infection and
reintroduction into the host can be achieved by known techniques (e.g., see
Dai, et al.,
1992, Proc. Natl. Acad. Sci. USA 89: 10892-10895).
To this end, in vivo direct injection into cells or tissue is an additional
method for
local delivery of the vector molecule encoding a CD2 binding agent. Direct
injection
30 ultimately results, upon expression of the gene product, in systemic
delivery of a
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-35-
therapeutic product-within the mammalian host (Wolff, et al, 1990, Science
247: 1465-
1468; Raz, et al., 1993. Proc. Natl. Acad. Sci. USA 90:4523-4527). Direct
injection of
naked DNA, preferably a non-viral vector such as plasmid DNA, is utilized in
this mode of
localized delivery.
Any eukaryotic promoter and/or enhancer sequence available to the skilled
artisan
which is known to up-regulate expression of the nucleic acid of interest may
be used in
plasmid vector constructions, including but not limited to a cytomegalovirus
(CMV)
promoter, a Rons Sarcoma virus (RSV) promoter, a Marine Leukemia Virus (MLV)
promoter, a beta -actin promoter, as well as any cell-specific eukaryotic
promoter sequence
1o that would be known to be active in the cell targeted for transduction.
Moreover, any of the
methods known in the art for the insertion of polynucleotide sequences into a
vector may
be used. See, for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and Ausubel et
al.,
Current Protocols in Molecular Biology, J. Wiley & Sons, NY ( 1992).
Conventional
is vectors consist of appropriate transcriptional/translational control
signals operatively linked
to the polynucleotide sequence for a particular interferon.
Promoters/enhancers may also be
used to control expression of interferons. (See Section III)
Expression vectors compatible with mammalian host cells for use in gene
therapy
of cells include, for example, plasmids; avian, marine and human retroviral
vectors;
2o adenovirus vectors; herpes viral vectors; parvoviruses; and non-replicative
pox viruses. In
particular, replication-defective recombinant viruses can be generated in
packaging cell
lines that produce only replication-defective viruses. See Current Protocols
in Molecular
Biology: Sections 9.10-9.14 (Ausubel et al., eds.), Greene Publishing
Associcates,1989.
Specific viral vectors for use in gene transfer systems are now well
established.
25 See for example: Madzak et al., J. Gen . Virol., 73: 1533-36 (1992)
(papovavitus SV40);
Berkner et al., Curr. Top. Microbiol. Immunol., 158: 39-61 (1992)
(adenovirus); Moss et
al., Curr. Top. Microbiol. Immunol., 158: 25-38 (1992) (vaccinia virus);
Muzyczka, Curr.
Top. Microbiol. Immunol., 158: 97-123 (1992) (adeno-associated virus);
Margulskee, Curr.
Top. Microbiol. Immunol., 158: 67-93 (1992) (herpes simplex virus (HSV) and
Epstein-
3o Barr virus (HBV)); Miller, Curr. To~Microbiol. Immunol., 158: 1-24 (1992)
(retrovirus);
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Brandyopadhyay et al., Mol. Cell. Biol., 4: 749-754 (1984) (retrovirus);
Miller et al.,
Nature, 357: 455-450 (1992) (retrovirus); Anderson, Science, 256: 808-813
(1992)
(retrovirus).
Preferred vectors arc DNA viruses that include adenoviruses (preferably Ad-2
or
Ad-5 based vectors), herpes viruses (preferably herpes simplex virus based
vectors), and
parvoviruses (preferably "defective" or non-autonomous parvovirus based
vectors, more
preferably adeno-associated virus based vectors, most preferably AAV-? based
vectors).
See, e.g., Ali et al., Gene Therayy l: 367-384,1994; U,S. Patent 4,797,368 and
x,399,346
- and discussion below. Adenovirus vectors are capable of providing extremely
high levels
of transgene delivery to virtually all cell types, regardless of the mitotic
stale. High titers
( 10' ~ plaque forming units/ml) of recombinant virus can be easily generated
in 293 cells
(an adenovirus-transformed, complementation human embryonic kidney cell line:
ATCC
CRL1573) and cryo-stored for extended periods without appreciable losses. The
efficiency
of this system in delivering a therapeutic transgene irr vivo that complements
a genetic
t5 imbalance has been demonstrated in animal models of various disorders. See
Y.
Watanabe, Atherosclerosis, 36: 261-268 (1986); K. Tanzawa et al, FEBS Letters,
118(1):81-84 (1980); J.L. Golasten et al. New Enel.J. Med., 309 (11983): 288-
296 (1983);
S. Ishibashi et al, J. Clin. Invest., 92: 883-893 ( 1993); and S. Ishibashi et
al, J. Clin. Invest.,
93: 1889-1893 (1994). Indeed. recombinant replication defective adenovirus
encoding a
cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved
for use
in several human CF clinical trials. See, e.g., J. Wilson, Nature, 365: 691-
692 (Oct., 21,
-- 1993). Further support of the safety of recombinant adenoviruses for gene
therapy is the
extensive experience of live adenovirus vaccines in human populations.
Adeno-associated viruses (AAV) have also been employed as vectors for somatic
gene therapy. AAV is a small, single-stranded (ss) DNA virus with a simple
genomic
organization (4.7 kb) that makes it an ideal substrate for genetic
engineering. Two open
reading frames encode a series of rep and cap polypeptides. Rep polypeptides
(rep78,
rep68, rep 62 and rep 40) are involved in replication, rescue and integration
of the AAV
genome. The cap proteins (VPI, VP2 and VP3) form the virion capsid. Flanking
the rep
3o and cap open reading frames at the 5' and 3' ends are 145 by inverted
terminal repeats
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(ITRs), the first 125-bp of which are capable of forming Y- or T-shaped duplex
structures.
Of importance for the development of AAV vectors, the entire rep and cup
domains can
be excised and replaced with a therapeutic or reporter transgene. See B.1.
Carter, in
Handbook of Parvoviruses, ed., P. Tijsser, CRC Press, pp. 155-168 (1990). It
has been
shown that the ITRs represent the minimal sequence required for replication,
rescue,
packaging, and integration of the AAV genome. High level gene expression fom
AAV in
mice was shown to persist for at least 1.5 years. See Xiao, Li and Samulski
(1996) Journal
of Virolosy 70, 8089-8108. Since there was no evidence of viral toxicity or a
cellular host
immune response, these limitations of viral gene therapy have been
overcome.Fisher et al.
t0 (Nature Medicine (1997) 3, 306-312) reported stable gene expression in mice
following
injection into muscle of AAV. Again, the virus was safe. No cellular or
humoral immune
response was detected against the virus or the foreign gene product.
Animal Models: The presence of suitable animal models of the various
indications
~5 (such as the well known EAE model for Multiple Sclerosis) allows testing of
the CD2
binding agents and the methods described. Several animal models are described
in the
Examples.
Deposits
2o Murine hybridoma cells and anti-LFA-3 antibodies useful in the present
invention
are exemplified by cultures deposited under the Budapest Treaty with American
Type
Culture Collection, Rockville, Maryland, U.S.A., on March 5, 199 1, and
identified as:
Designation ATCC Accession Number
2~ 1 E6 HB I 0693
HC- IB 11 HB 10694
7A6 HB 10695
8B8 HB 10696
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A bacteriophage carrying a plasmid encoding transmembrane LFA-3 was deposited
under the Budapest Treaty with In Vitro International, Inc., Linthicum,
Maryland, U.S.A.,
on May 28, 1987 under Accession No. IVI-10133. This deposit was transferred on
June
20,1991 to the American Type Culture Collection ( 10801 University Blvd.,
Mantissas,
s Virginia 20110-2209, United States) and identified as:
Designation ATCC Accession No.
AHT 16[hgt 10/LFA-3] 75107
E. coli transformed with a plasmid encoding PI-linked LFA-3 was deposited
under
the Budapest Treaty with In Vitro International, Inc. on July 22, 1988 under
Accession No.
IV1-10 1 80. This deposit was transfened to American Type Culture Collection
(present
address 1080 University Blvd., Mantissas, Virginia. U.S.A.) on June 20, 1991
and
identified as:
Designation ATCC Accession No. '
~5 p24 68788
Sequences
The following is a summary of the sequences set forth in the Sequence Listing:
2o SEQ )D NO: 1 DNA sequence of transmembrane LFA-3
SEQ ID N0:2 Amino acid sequence of transmembrane LFA-3
SEQ m N0:3 DNA sequence of PI-linked LFA-3
SEQ ID N0:4 Amino acid sequence of PI-linked LFA-3
SEQ )D NO:S DNA sequence of CD2
25 SEQ m N0:6 Amino acid sequence of CD2
SEQ ID N0:7 DNA sequence of LFA3TIP
SEQ ID NO: 8 Amino acid sequence of LFA3T1P
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In order that this invention may be better understood, the following examples
are
set forth. These examples are for purposes of illustration only, and are not
to be construed
as limiting the scope of the invention in any manner.
EXAMPLE 1: Modulation of Memory Effector T lymphocytes
This example illustrates that a particular CD2 binding agent, LFA3 TIP (SEQ ID
NO: 8), will selectively modulate memory effector T lymphocytes in mammalian
subjects
with psoriasis.
Materials and Methods
t0 We performed a Phase Il, randomized, double-blind, parallel design, four
arm,
placebo-controlled investigational trial of LFA3TIP in patients with chronic
plaque
psoriasis. The primary study objective was to determine the relationship of
clinical
response to dosages ranging from 0.025 to 0.15 mg/l:g body weight. At 22 sites
in the
United States, 229 subjects ranged 18 to 70 years were enrolled. Entry
criteria included
t5 plaque psoriasis of at least one year duration, with greater than 10%
surface area
involvement and not controlled by topical agents: The mean duration of disease
was 16
years (1-63 year range). Phototherapy and systemic therapy was discontinued
one month
prior to the start of the study. LFA3TIP is packaged as a frozen solution in
vials containing
mg/mL fusion protein per vial and exipient materials (sodium chloride,
monobasic
potassium phosphate, and dibasic sodium phosphate). The vials are stored at -
70°C (-94°F)
and thawed in the refrigerator or at room temperature and diluted with 0.9%
sodium
chloride to a final volume of 5 mL for IV bolus administration.
The CD2 binding agent was administered once a week by i.v. bolus for 12 weeks;
subjects were followed for an additional 12 weeks after the last dose. Disease
severity was
assessed using a Physicial Global Assessment and the Psoriasis Activity and
Severity Index
(PASI) with the primary efficacy endpoints assessed at 2 weeks following the
last dose of
the study drug.
Peripheral blood was drawn from patients and lymphocytes separated from using
ficoll-hypaque gradient centrifugation. Cells (about 3 x 105 ) were assayed by
flow
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cytometry (FACSTAR PLUS, Becton Dickinson, San Jose, CA). Ten thousand events
were
monitored.
Results
We see statistically significant differences between LFA3TIP and placebo in a
number of endpoints, including reduction in PASI score in the two higher
LFA3TIP
dosages. Clinical response rates increased with greater concentration of
LFA3TIP (data not
presented). Moreover, treatment with this CD2 binding agent was associated
with a
reduction in peripheral CD4+ and CD8+ T lymphocytes and to natural killer
cells. We
observed selective reduction of CD45 RO+ memory-effector T cells over CD45RA+
naive
CD4+ and CD8+ cells. Specifically, flow cytometric analysis revealed a greater
reduction
in high density T cells with memory/effector phenotypes (CD4-CD45R0 and CD8-
CD45R0) than in low density CD2 T cells with naive phenotypes (CD4-CD45RA and
CD8-CD45RA). Figure I is a graph showing the selective reduction of memory
effector
T cells relative to naive T cells in patients with psoriasis receiving
treatment with
LFA3TIP. The horizontal bar above the X-axis represents the duration of
chronic
treatment, which was stopped at approximately 80 days. The curves represent
the mean
absolute lymphocyte subset counts from the 57 patients in each treatment
group. There are
statistically significant dose-related reductions in memory effector T cells
(CD 45 RO+).
No changes in peripheral naiive T cells (CD45 RA+) were observed (CD45 RA+)
regardless of treatment regimen. T cell counts in the various CD2 binding
agent treatments
show a decrease, approaching some relatively constant value, during the 80 day
chronic
administration period. After treatment was terminated, the memory effector T
cells count
began to increase again.
LFA3TIP was well tolerated with non drug-related serious adverse events
reported.
A transient, mild, dose-dependent reduction from pro-infusion levels in the
absolute
number of peripheral lymphocytes was observed in all subjects who received
LFA3TIP.
This reduction was noted in total, CD2, CD4, and CD8 lymphocytes, but not in
CD19
lymphocytes.
EXAMPLE 1I: Treatment Of Rheumatoid Arthritis
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Human patients having rheumatoid arthritis (RA) are selected for treatment
using a
CD2 binding agent such as LFA3-TIP. This material is prepared and packaged in
Example
1 as described and is administered to patients at doses of about 7.5 to 10 mg
weekly for a
period of 12-24 weeks. To determine optimum dose and schedule, a parallel
design study
using the above-referenced different regimens are performed. Peripheral blood
is drawn
from patients and lymphocytes separated from using ficoll-hypaque gradient
centrifugation.
Cells (about 3 x 10' ) are assayed by flow cytometry (FACSTAR PLUS, Becton
Dickinson,
San Jose, CA), as above. Patients are monitored using several indicia,
including joint
swelling and tenderness scores, that are included as part of the standardized
"ACR 20"
t0 studies. See Felson, D.T. et al., "American College of Rheumatology Core
Set of Disease
Activity Measurements for Rheumatoid Arthritis Clinical Trials". Arth. Rheum.,
26: 729-
740 ( 1993).
Example III: Treatment of Inflammatory Bowel Disease (IBD), Crohn's Disease
t5 and Colitis
Inflammatory bowel disease is a generic term relating to a group of chronic
inflammatory disorders of unknown etiology involving the gastrointestinal
tract.
Ulcerative colitis (UC) and Crohn's disease, the two major forms of idiopathic
Inflammatory Bowel Disease (IBD) in humans, are widespread and poorly
understood
2o disorders (Kirsner, J. B., et al., eds., Inflammatory Bowel Disease: 3rd
ed., Lea and
Febiger, Philadelphia ( 1988); Goldner, F. H., et al., Idiopathic Inflammatory
Bowel
Disease, in Stein, J. H., ed., Internal Medicine, Little Brown & Co., Boston,
pp. 369-380
(1990); Cello,1. P., et al.. Ulcerative Colitis, in Sleisenger, M. H., et al..
eds.,
Gastrointestinal Disease: Pathophysiology Diagnosis Management, W. B. Saunders
Co.,
25 Philadelphia, p. 1435 ( 1989)). While the cause of inflammatory bowel
disease is unknown,
possible etiologic factors include infectious, immunologic, familial or
psychological
factors. Secondary extraintestinal manifestations such as arthritis and
pericholangitis often
times occur during chronic inflammatory bowel disease. Drug therapy of
inflammatory
bowel disease includes administration of anti-inflammatory drugs,
sulfasalazine (the active
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compound thought to be 5-aminosalicylate, most likely by inhibiting
prostaglandin
synthesis) and glucocorticoids.
Crohn's Disease or Regional lleitis, is the term applied to a condition in
which
there is an inflammation of an area of the small intestine. This is usually
accompanied by
colicky abdominal pain, irregularity of the bowels, loss of weight and slight
fever. The
abdomen is generally distended and the thickened intestine may be felt. The
narrowed
intestinal canal may become obstructed, necessitating immediate operation. The
cause of
the disease is unknown. The primary lesion is hyperplasia of the lymph tissue
in the
submucosa of the intestine and in the lymph glands. Crohn's Disease is a
chronic condition
to of the gastrointestinal tract and infects most commonly the ileum, colon or
a combination
of both. It is distinguished from ulcerative colitis by a differential
diagnosis.
The pathology of ulcerative colitis usually refers to a more superficial
mucosal
disease in contrast to Crohn's disease with its deep, often transmucosal
involvement and
fissures (Riddell, R. H., ed., Pathology of Drug-induced and Toxic Diseases,
Churchill
~5 Livingstone, New York (1982); Morrison. B. C., et al.. eds.,
Gastrointestinal Pathology, 2d
ed., London ( 1979); Fenoglio-Preiser, C. M., et al., eds., Gastrointestinal
Pathology: An
Atlas and Text, Raven Press, New York ( 1989); Goldman, H., et al., Hum.
Pathol. 13:98 I -
1012 ( 1982)). Ulcerative colitis typically involves the rectum and extends
proximally
without intervening uninvolved "skip" areas which are usually the hallmarks of
Crohn's
2o disease.
The available animal models for IBD can be divided into naturally occurring
and
experimentally-induced animal models. Unfortunately, only a few spontaneous
and rarely
occurring models of intestinal inflammation due to a genetic defect are
available and most
of these are not idiopathic but are induced by bacteria or other infectious
agents (e.g.,
25 hyperplasia, crypt abscesses, ulcers in mice with Bacillus psyliformnis and
hamster with
"rod-shaped bacteria") (Strober, W., Die. Dis: Sci. 33 Suppl.:3S-lOS ( 1988)).
Rare forms
of spontaneous ulcerative colitis and granulomatous enterocolitis also occur
in rats and
horses, respectively. In addition, marmosets and the cotton-top tamarin are
used as a
spontaneous model of ulcerative colitis and colonic adenocarcinomas (Clapp, N.
K., et al.,
3o eds., Dig. Dis. Sci. 33 Suppl.:lS-1585 (1988)). Experimentally induced
animal models of
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ulcerative colitis are usually produced by exposure to toxic dietary
substances,
pharmacologic agents or other environmental chemicals, or by administration of
materials
derived from patients, or by manipulation of the animal's immune system
(Strober, W.,
Dig. Dis. Sci. 33 Suppl.:3S-lOS (1988); Beekan, W. L., Experimental
inflammatory bowel
disease, in: Kirsner, J. B., et al., eds., Inflammatory Bowel Disease, Lea and
Febiger,
Philadelphia, pp. 37-49 ( 1988); Onderdonk, A. B., Dig. Dis. Sci. 33
Supp1.:405-44S
( 1988)).
One can examine the potential therapeutic efficacy and mechanisms of action of
a
CD2 binding agent in the treatment of Crohn's disease using a cotton-top
tamarin model.
See, for example, J. Madara et al., "Characterization of Spontaneous Colitis
in Cotton-Top
Tamarin (Saguinus Oedipus) and lts Response to Sulfasalazine,"
Gastroenterolooy, 88, pp.
13-19 (1985). A stock solution in sterile saline of CD2 binding agent (e.g.,
LFA3TIP) and
a placebo control (saline only), are prepared for administration to ten cotton
top tamarins
(CTTs) exhibiting symptoms of spontaneous colitis (i.e., diarrhea, etc.; see,
Madara et al.,
supra). Five CTTs receive CD2 binding agent and five receive placebo, by
intravenous
injection. The CTTs receiving CD2 binding agent are injected with 1 mg agent
per day
(i.e., about 2 mg/kg/day, based on approximate half-kilogram weight of a CTT)
for eight
days (on Days 0, 1, 2, 3, 4, 5, 6, and 7 of the trial). Colon tissue samples
obtained from the
animals are biopsied every other day (on Days 0, 2, 4, 6, 8, and 10 of the
trial). Data from
the biopsies are used to determine an acute inflammation index for each
animal, giving a
semi-quantitative analysis of the course of the colitis. (See, Madara et al
supra.) Peripheral
blood is drawn and lymphocytes separated from using ficoll-hypaque gradient
centrifugation. Cells (about 3 x 105 ) are assayed by flow cytometry (FACSTAR
PLUS,
Becton Dickinson, San Jose, CA), as above. Results are expected to show
selective
reduction of memory effector T cells and show that treatment with CD2 binding
agent
results in a significant (p < 0.01) decrease in acute inflammation index.
In humans, patients having IBD are selected for treatment using a CD2 binding
agent such as LFA3-TIP. This material is prepared and packaged in Example I as
described and is administered to patients at doses of about 7.5 to 10 mg
weekly for a
period of 1?-24 weeks. To detemune optimum dose and schedule, a parallel
design study
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using the above-referenced different regimens arc performed. Peripheral blood
is drawn
from patients and lymphocytes separated from using ficoll-hypaque gradient
centrifugation.
Cells (about 3 x 105 ) are assayed by flow cytometry (FACSTAR PLUS, Becton
Dickinson,
San Jose, CA), as above. Patients are monitored using several indicia,
including the
Crohn's Disease Activity Index (Kjeldsen,J. and Schaffalitzky de Muckadell,
O.B., Scand.
J. Gastroenterol. 28: 1-9 (1993).
Example IV: Treatment of Uveitis
Uveitis is a disease of the eye which can be located throughout the eye
including the
l0 posterior and anterior chambers of the eye as well as the vitreous body.
Uveitis, the
inflammation of the uvea, is responsible for about 10% of the visual
impairment in the
United States. Panuveitis refers to inflammation of the entire uveal
(vascular) layer of the
eye. Posterior uveitis generally refers to chorioretinitis and anterior
uveitis refers to
iridocyclitis. The inflammatory products, that is, cells, fibrin, excess
protein, of these
inflammations are commonly found in the fluid spaces of the eye including the
anterior
chamber, posterior chamber and vitreous space as well as the tissue involved
in the
inflammatory response. Uveitis may occur following surgical or traumatic
injury to the eye,
as a component of an autoimmune disorder such as rheumatoid arthritis,
Behcet's disease,
ankylosing spondylitis, sarcoidosis, as an isolated immune mediated ocular
disorder, i.e.,
pars planitis, iridodyclitis etc., unassociated with known etiologies, and
following certain
systemic diseases which cause antibody-antigen complexes to be deposited in
uveal tissues.
Together these disorders represent non-infectious uveitities.
Treatment in Mammals
Three cynomologous monkeys are used. Animals are anesthetized using ketamine
(30 mg/kg) for all procedures. Devices containing 6 mg of LFA3-TIP are
implanted into
the right eye and control devices consisting of polymer alone are implanted
into the left eye
as described. The monkey has a well developed pans plana so cryopexy is
unnecessary. All
devices are implanted 3 mm posterior to the limbus in the inferior, temporal
quadrant.
Animals are anesthetized at 1 week, 2 weeks, 4 weeks, 2 months, 4 months, and
6 months
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and undergo examination with indirect ophthalmoscopy and electroretinography.
Electroretinography is performed as described except that averages of 5
responses are
acquired with a Nicolet CA-1000 clinical averager (Madison, Wis.) and a CKA
Ganzfeld
flash (Gaithersburg, Md.). At 6 months a sample of blood is analyzed by HPLC
for
cyclosporine (detection limit = ?5 ng/ml). After 6 months the animals are
sacrificed with
an overdose of pentobarbital and then were immediately perfused via the left
ventricle with
10% buffered formalin. Eyes are placed in fixative and then subsequently
embedded in
paraffin, sectioned and examined in a masked fashion for evidence of toxicity.
Peripheral
blood was drawn and lymphocytes separated from using ficoll-hypaque gradient
1o centrifugation. Cells (about 3 x 105 ) were assayed by flow cytometry
(FACSTAR PLUS,
Becton Dickinson, San Jose, CA).
Pharmacokinetics
Twenty four animals (Group 1 ) were used. CD2 binding agent is mixed into
stock
solutions of 50:50 ethanol:water at a concentration of 1 ug/20 ul and 10 ug/20
ul. Tritiated
CD2 binding agent ( 1 uCi/20 ml) was also added to the stock solution. Animals
are
anesthetized with ketamine (60 mg) and xylazine (20 mg) and the pupils are
dilated with
one drop of phenylephrine hydrochloride (2.5%) and tropicamide ( 1 %). An
intravitreal
injection of CD2 binding agent is then given through the superior rectus
muscle
2o approximately 5 mm posterior to the limbus using a 30 gauge needle. Twelve
animals
receive a dose of 1 ug in 20 ul and 12 animals received a dose of 10 ug in 20
ul). The
injection is given into the midvitreous cavity and care is taken to avoid the
lens. For the 1
ug group, 3 animals are sacrificed at 0.5, 3, 6 and 24 hours. For the 10 ug
group, 3 animals
are sacrificed at 0.5, 6, 24 and 48 hours. The eyes are enucleated and then
immediately
frozen at - 70o C. Tissues are prepared for assay as described below.
Intravitreal half-life
and volume of distribution are determined from regression analysis of a 1 n
concentration
versus time plot.
Treatment
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Eleven animals are used. CD2 binding agent contained in a sustained release
device is
inserted into the right eye. A device consisting of polymers only is inserted
into the left eye.
Prior to device insertion an examination consisting of ophthalmoscopy and
electroretinography (ERG) was performed. Scotopic and photopic
electroretinograms
s (ERG'S) is recorded from both eyes using contact lens electrodes (ERG-Jet)
with a two-
channel clinical signal averager (Cadwell 5200, Kennewick, Wash.) and a
Ganzfeld flash
unit (Cadwell VPA-10, Kennewick, Wash.). Dark adapted ERGS, performed after at
least
30 minutes of dark adaptation, are elicited at 0.33 Hz and 20 stimulus
presentations are
averages. Light adapted ERGS were performed at 2.8 Hz after at least 5 minutes
of light
1o adaptation and are also the average of 20 stimuli. Resultant waves are
evaluated for
amplitude and latency. To minimize the effect of individual and daily
variation ( 13-15) the
ERG responses are evaluated using a ratio of the amplitude of the experimental
(right) to
the amplitude of the control (left). When the amplitudes of the experimental
and control
eyes are equal, the ratio equals 1. A decrease in the ratio reflects a
relative decrease in the
I5 amplitude of the experimental eye.
Examinations are repeated on days 1, 7, 14 and then every two weeks for the
next 6
months. Three animals are sacrificed at 6 weeks and two animals are sacrificed
at 16
weeks. To assess for reversible toxicity the device is removed from 3 animals
at 16 weeks.
These animals are examined weekly for two months and then sacrificed. The
remaining
20 two animals are sacrificed at 26 weeks. Following sacrifice, the animals
are perfused with
fixative (either 10% buffered formalin or 6% phosphate buffered
glutaraldehyde) via the
left ventricle. The eyes are enucleated and placed in fixative. Three 3 mm by
6 mm
specimens from the posterior pole are then embedded in either paraffin or
plastic and
sectioned. One section from each specimen is examined in a masked manner for
evidence
25 of toxicity.
EXAMPLE V. Treatment of Psoriatic Arthritis
This example provides a protocol to test the clinical effects and tissue
response of a
CD2 binding agent in patients with psoriatic arthritis.
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Psoriatic arthritis occurs in 5-8% of patients with psoriasis and is a common
chronic rheumatic disease that may result in considerable joint damage if left
untreated.
Recent clinical studies have focused on the identification of poor prognostic
factors. Early
aggressive treatment is indicated in patients with significant joint
inflammation and certain
HLA antigens. Psoriasis and psoriatic arthritis are considered typical T cell
mediated
diseases. lmmunopathogenic evidence continues to point toward an HLA class I
mediated
disease with perhaps an important role for CD8+ T cells.
STUDY DESIGN
This is a Phase II pilot study of an exemplary CD2 binding agent in patients
with
psoriatic arthritis.
The study will consist of a 3 month treatment period with weekly intravenous
injections of
a CD~'. binding agent. The effects on psoriatic skin lesions and on arthritis
will be
measured by clinical and routine laboratory methods at the start. during and
after the
treatment period. Directly preceding, after 4 weeks and following the
treatment period
synovial biopsies will be taken for immunohistologic investigations using
standardized
methods. Preceding, after 4 and 12 weeks and at the end of a 3 months post-
treatment
period skin biopsies will be taken for immunohistologic investigations using
standardized
methods.
STUDY POPULATION
Number of Subjects
Patients with psoriatic arthritis are screened in order to select patients for
the study if all of
the following inclusion criteria are met:
1 ) Male or female patients between 18 and 70 years, inclusive.
2) Female patients must be of non-childbearing potential (surgically sterile
or at least one
year post-menopausal) of have a negative pregnancy test at the screening visit
and
maintain an effective birth control method at least one month prior to study
drug
administration, during study and in a period cf 6 months post-,dosing.
3) Patients must fulfil the criteria for diagnosis of definite psoriatic
arthritis
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4) Patients must have clinically apparent arthritis of at least one knee joint
and 3 other
joints, morning stiffness of at least 30 minutes and an erythrocyte
sedimentation rate
(ESR) of at least 28 mm.
5) Patients must have a diagnosis of plaque-type psoriasis greater then 12
months prior to
the first dose of study drug.
6) Patients must have a PASI of 12 or greater.
7) Patients must have discontinued all immunomodulating medication such as
methotrexate, cyclosporin and corticosteroids and all other compounds used to
treat
psoriasis and/or psoriatic arthritis except non steroidal anti-inflammatory
drugs
(NSAms), at least four weeks prior to the Screening Visit
8) Patients must have an absolute CD4+ lymphocyte count at or greater than the
lower
limit of normal within 14 days prior to the first dose.
Treatment Schedule
The CD2 binding agent will be administered as an intravenous bolus of 7.5 mg
fixed dose, given once every week for a total of twelve doses.
Modification of Treatment Schedule
In order for a subject to receive the next dose of study drug, the following
must occur:
20 - The absolute number of peripheral lymphocytes obtained for each subject
at the site
within 24 hours of administration, must be greater than 67% of the lower limit
of
normal OR greater than 50% of the subject's baseline measure.
- The absolute CD4+ lymphocytes obtained prior to the previous dose must be
greater
than 300 cells/mm'.
If any one of the above criteria is not met, the subject's dose will be
withheld and the
subject will return the following week for repeat evaluation. If any subject
experiences a
sustained reduction in absolute number of peripheral lymphocytes or absolute
CD4+
lymphocytes for more than 28 days, study drug will be permanently
discontinued.
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Discontinuation of Study Drug and Subjects
The following sections describe the criteria for discontinuing study drug, and
the criteria
for withdrawing from the study.
SCHEDULE OF EVENTS
Tests and Evaluations
All blood samples for hematology and lymphocyte subset analysis will be
collected at the
same time of the day in order to prevent artifact of diurnal variation.
1o The following tests and evaluations are to be conducted at the time points
indicated prior to
each dose of study drug, unless. otherwise specified:
- A complete physical examination to be performed at Visits 5, 9, and 13.
- Measurement of vital signs to be performed at Visits 1 through 13 and Visits
15
through 17.
- Recording of body weight to be performed at Visits 1 through 13 and Visits
15 through
17.
- Urinalysis to be performed at Visits 5, 9, 13 and 17.
- Blood chemistry to be performed at Visits 5, 9, 13 and 17.
- Hematology to be performed at Visits 1 through 17. The first lab test to be
performed
2o within 48 hours prior to the first dose of study drug. All other tests to
be performed
within 24 hours prior to each dose.
- Lymphocyte subset analysis to be performed at Visits 1 through 17.
- Pregnancy (urine) test for women to be performed at Visits 5, 13 and 17.
- Quantitative measurement of immunoglobulins IgG, IgA, and IgM to be
performed at
?5 Visit 13.
- PASI to be performed at Visits 1, 3, 5, 7, 9, 11 and 13 through 17.
- Nail evaluation to be performed at Visits 1, 3, 5. 7, 9, 11 and 13 through
17.
- Measuring of s1L-2R and sCD27 at Visits 1, 3. 5, 7, 9, 11 and 13 through 17.
- Skin punch biopsy of involved, non-target lesion from an area not exposed to
sunlight
3o to be performed at Visits 1, 5, 13 with an optional biopsy at Visit 17.
CA 02514899 1999-08-31
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- ACR Core Set to be evaluated at Visits 1, 5, 9, 13 and 17.
- ACR Improvement Criteria to be evaluated at Visits 5, 9, 13 and 17.
- Knee arthroscopic biopsy of synovium to be performed at Visits -l, 5 and 13.
EFFICACY ASSESSMENTS
Clinical Efficacy Assessments
Arthritis
All efficacy assessments on arthritis are to be performed by the same
investigator/rheumatologist for each subject:
lU - ACR Core Set and Improvement Measurements
Skin lesions
All efficacy assessments on skin lesions are to be performed by the same
investigator/dermatologist for each subject: '
- PASI score
t5 - Nail evaluation
Laboratory Efficacy Assessments
- Arthroscopic synovial biopsy
- Immunohistological studies of synovium
- Serum disease activity markers for psoriasis
20 - Skin biopsy
- Immunohistological studies of the skin
SAFETY ASSESSMENTS
Clinical Safety Assessments
25 - Physical examination.
- Vital signs (temperature, heart rate, respiration rate, and supine blood
pressure).
- Body weight.
- Monitoring for infection.
- Monitoring adverse events.
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-51-
Laboratory Safety Assessments
- Hematology: complete blood count with differential and platelet count will
be
performed within 24 hours of dosing and monitored by the investigator at each
site.
- Blood chemistry profile: sodium, potassium, chloride, bicarbonate, blood
urea nitrogen,
creatinine, calcium, phosphate, albumin, total protein, alkaline phosphatase,
total
bilirubin, ALAT, ASAT, and gamma glutamyl transaminase.
- Urinalysis.
to Product/Trial Specific Safety Assessments
- Peripheral lymphocyte subset quantitation using flow cytometry with well
defined
subset markers for T lymphocytes (CD4, CD8).
STATISTICAL STATEMENT AND ANALYTICAL PLAN
~5 Sample Size Considerations
A pilot study with 10 subjects is considered appropriate for evaluation of
safety and
efficacy. Past experience with CD2 binding agents such as LFA-3/IgG, fusion
protein has
demonstrated consistent effects on total lymphocytes, and CD4 and CD8 subsets.
2o Description of Endpoints
The primary clinical efficacy endpoints for arthritis and psoriasis are based
on the end of
treatment after 12 weeks.Surrogate endpoints for clinical efficacy are based
on 4 weeks and
8 weeks of treatment. The primary safety endpoint is based on the end of a
post treatment
observation period of 3 months. Surrogate endpoints for immunohistological
changes in
25 skin and synovium are based on two points in time: after 4 weeks and after
12 weeks of
treatment.
Statistical Methods To Be Used in Objective Analyses
The general method of analysis will be to establish differences of clinical
and laboratory
30 parameters between baseline and the end of therapy. This will be done
using, for example,
CA 02514899 1999-08-31
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a one-way analysis of variance or covariance, or logistic regression,
Responses may need to
be transformed prior to analysis. Non-parametric techniques will be used when
appropriate.
Clinical Efficacy Analyses
The proportion of subjects demonstrating 2090 improvement in ACR Core Set
Measurements will be determined. The proportion of subjects with at least a
5090 decrease
from baseline in their PASI score will be determined. Other efficacy measures
include
minimum PASI score, total erythema score, total induration score, total
desquamation
score and nail score.
Laboratory Efficacy Analyses
Immunohistological studies of synovium and skin and serum levels of psoriasis
activity
markers will provide data that will be compared with baseline values.
While we have described a number of embodiments of this invention, it is
apparent
~5 that our basic embodiments can be altered to provide other embodiments chat
utilize the
processes of this invention. Therefore, it will be appreciated that the scope
of this
invention includes all alternative embodiments and variations which are
defined in the
foregoing specification and by the claims appended hereto; and the invention
is not to be
limited by the specific embodiments that have been presented herein by way of
example.
CA 02514899 1999-08-31
1
SEQUENCE LISTING
<110> Biogen, Inc.
<120> Method of Modulating Memory Effector T-Cells and
Compositions
<130> Seq 1-8
<140> PCT US 99/20026
<141> 1999-08-31
<160> 8
<170> PatentIn Ver. 2.0
<210> 1
<211> 753
<212> DNA
<213> Homo Sapiens
<400> 1
atggttgctg ggagcgacgc ggggcgggcc ctgggggtcc tcagcgtggt ctgcctgctg 60
cactgctttg gtttcatcag ctgtttttcc caacaaatat atggtgttgt gtatgggaat 120
gtaactttcc atgtaccaag caatgtgcct ttaaaagagg tcctatggaa aaaacaaaag 180
gataaagttg cagaactgga aaattctgaa ttcagagctt tctcatcttt taaaaatagg 240
gtttatttag acactgtgtc aggtagcctc actatctaca acttaacatc atcagatgaa 300
gatgagtatg aaatggaatc gccaaatatt actgatacca tgaagttctt tctttatgtg 360
cttgagtctc ttccatctcc cacactaact tgtgcattga ctaatggaag cattgaagtc 420
caatgcatga taccagagca ttacaacagc catcgaggac ttataatgta ctcatgggat 480
tgtcctatgg agcaatgtaa acgtaactca accagtatat attttaagat ggaaaatgat 540
cttccacaaa aaatacagtg tactcttagc aatccattat ttaatacaac atcatcaatc 600
attttgacaa cctgtatccc aagcagcggt cattcaagac acagatatgc acttataccc 660
ataccattag cagtaattac aacatgtatt gtgctgtata tgaatggtat tctgaaatgt 720
gacagaaaac cagacagaac caactccaat tga 753
<210> 2
<211> 249
<212> PRT
<213> Homo Sapiens
<400> 2
Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val
1 5 10 15
Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln
20 25 30
Ile Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val
35 40 45
Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu
50 55 60
Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg Val
65 70 75 80
CA 02514899 1999-08-31
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Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr Ser
85 90 95
Ser Asp Glu Asp GIu Tyr Glu Met Glu Sex Pro Asn Ile Thr Asp Thr
100 105 110
Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr Leu
115 120 125
Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile Pro
130 135 140
Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp Cys
145 150 155 160
Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys Met
165 170 175
Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro Leu
180 185 190
Phe Asn Thr Thr Ser Ser Ile Ile Leu Thr Thr Cys Ile Pro Ser Ser
195 200 205
Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala Val
210 215 220
Ile Thr Thr Cys Ile VaI Leu Tyr Met Asn Gly Ile Leu Lys Cys Asp
225 230 235 240
Arg Lys Pro Asp Arg Thr Asn Ser Asn
245
<210> 3
<211> 723
<212> DNA
<213> Homo Sapiens
<400> 3
atggttgctg ggagcgacgc ggggcgggcc ctgggggtcc tcagcgtggt ctgcctgctg 60
cactgctttg gtttcatcag ctgtttttcc caacaaatat atggtgttgt gtatgggaat 120
gtaactttcc atgtaccaag caatgtgcct ttaaaagagg tcctatggaa aaaacaaaag 180
gataaagttg cagaactgga aaattctgaa ttcagagctt tctcatcttt taaaaatagg 240
gtttatttag acactgtgtc aggtagcctc actatctaca acttaacatc atcagatgaa 300
gatgagtatg aaatggaatc gccaaatatt actgatacca tgaagttctt tctttatgtg 360
cttgagtctc ttccatctcc cacactaact tgtgcattga ctaatggaag cattgaagtc 420
caatgcatga taccagagca ttacaacagc catcgaggac ttataatgta ctcatgggat 480
tgtcctatgg agcaatgtaa acgtaactca accagtatat attttaagat ggaaaatgat 540
cttccacaaa aaatacagtg tactcttagc aatccattat ttaatacaac atcatcaatc 600
attttgacaa cctgtatccc aagcagcggt cattcaagac acagatatgc acttataccc 660
ataccattag cagtaattac aacatgtatt gtgctgtata tgaatggtat gtatgctttt 720
taa 723
<210> 4
<211> 240
<212> PRT
<213> Homo Sapiens
CA 02514899 1999-08-31
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<400> 4
Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val
1 5 10 15
Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln
20 25 30
Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Sex Asn
35 40 45
Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala
50 55 60
Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg
65 70 75 80
Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr
85 90 95
Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp
100 105 110
Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr
115 120 125
Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile
130 135 140
Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp
145 150 155 160
Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys
165 170 175
Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro
180 185 190
Leu Phe Asn Thr Thr Ser Ser Ile Ile Leu Thr Thr Cys Ile Pro Ser
195 200 205
Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu AIa
210 215 220
VaI Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Met Tyr Ala Phe
225 230 235 240
<210> 5
<211> 1056
<212> DNA
<213> Homo Sapiens
<400> 5
atgagctttc catgtaaatt tgtagccagc ttccttctga ttttcaatgt ttcttccaaa 60
ggtgcagtct ccaaagagat tacgaatgcc ttggaaacct ggggtgcctt gggtcaggac 120
atcaacttgg acattcctag ttttcaaatg agtgatgata ttgacgatat aaaatgggaa 180
aaaacttcag acaagaaaaa gattgcacaa ttcagaaaag agaaagagac tttcaaggaa 240
aaagatacat ataagctatt taaaaatgga actctgaaaa ttaagcatct gaagaccgat 300
gatcaggata tctacaaggt atcaatatat gatacaaaag gaaaaaatgt gttggaaaaa 360
atatttgatt tgaagattca agagagggtc tcaaaaccaa agatctcctg gacttgtatc 420
CA 02514899 1999-08-31
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aacacaaccc tgacctgtga ggtaatgaat ggaactgacc ccgaattaaa cctgtatcaa 480
gatgggaaac atctaaaact ttctcagagg gtcatcacac acaagtggac caccagcctg 540
agtgcaaaat tcaagtgcac agcagggaac aaagtcagca aggaatccag tgtcgagcct 600
gtcagctgtc cagagaaagg tctggacatc tatctcatca ttggcatatg tggaggaggc 660
agcctcttga tggtctttgt ggcactgctc gttttctata tcaccaaaag gaaaaaacag 720
aggagtcgga gaaatgatga ggagctggag acaagagccc acagagtagc tactgaagaa 780
aggggccgga agccccacca aattccagct tcaacccctc agaatccagc aacttcccaa 840
catcctcctc caccacctgg tcatcgttcc caggcaccta gtcatcgtcc cccgcctcct 900
ggacaccgtg ttcagcacca gcctcagaag aggcctcctg ctccgtcggg cacacaagtt 960
caccagcaga aaggcccgcc cctccccaga cctcgagttc agccaaaacc tccccatggg 1020
gcagcagaaa actcattgtc cccttcctct aattaa 1056
<210> 6
<211> 351
<212> PRT
<213> Homo sapiens
<400> 6
Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn
1 5 10 15
Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu
20 25 30
Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe
35 40 45
Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp
50 55 60
Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu
65 70 75 80
Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His
85 90 95
Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr
100 105 110
Lys GIy Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu
115 120 125
Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu
130 135 140
Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln
145 150 155 160
Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp
165 170 175
Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val
180 185 190
Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu
195 200 205
Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met
210 215 220
CA 02514899 1999-08-31
S
Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln
225 230 235 240
Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val
245 250 255
Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln Ile Pro Ala Ser Thr
260 265 270
Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His
275 280 285
Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val
290 295 300
Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val
305 310 315 320
His Gln GIn Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys
325 330 335
Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn
340 345 350
<2I0> 7
<211> 1050
<212> DNA
<213> Homo sapiens
<400> 7
atggttgctg ggagcgacgc ggggcgggcc ctgggggtcc tcagcgtggt ctgcctgctg 60
cactgctttg gtttcatcag ctgtttttcc caacaaatat atggtgttgt gtatgggaat 120
' gtaactttcc atgtaccaag caatgtgcct ttaaaagagg tcctatggaa aaaacaaaag 180
gataaagttg cagaactgga aaattctgaa ttcagagctt tctcatcttt taaaaatagg 240
gtttatttag acactgtgtc aggtagcctc actatctaca acttaacatc atcagatgaa 300
gatgagtatg aaatggaatc gccaaatatt actgatacca tgaagttctt tctttatgtc 360
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 420
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 480
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 540
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 600
cgggtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 660
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 720
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 780
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 840
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 900
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1020
ctctccctgt ctccgggtaa atgagtgcgg 1050
<210> 8
<211> 347
<212> PRT
<213> Homo sapiens
<400> 8
Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val
1 5 10 15
CA 02514899 1999-08-31
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Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln
20 25 30
Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn
35 40 45
Val Pro Leu Lys Giu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala
50 55 60
Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg
65 70 75 80
Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr
85 90 95
Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp
100 105 110
Thr Met Lys Phe Phe Leu Tyr Val Asp Lys Thr His Thr Cys Pro Pro
115 120 125
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
130 135 140
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
145 150 155 160
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
165 170 175
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
180 185 190
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
195 200 205
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
210 215 220
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
225 230 235 240
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
245 250 255
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
260 265 270
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
275 280 285
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
290 295 300
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
305 310 315 320
CA 02514899 1999-08-31
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Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
325 330 335
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
