Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR MODULATING T CELL ACTIVATION
AND USES THEREOF
Technical Field
This invention relates generally to the field of immunology or
neuroimmunology.
In particular, the invention provides a method for reducing or inhibiting T
cell activation,
which method comprises administering an effective amount of an antagonist of
NCAM L1
to a mammal, wherein reduction or inhibition of T cell activation is
desirable, thereby
reducing or inhibiting T cell activation in said mammal. Combinations and
combinatorial
methods for modulating T cell activation are further provided. The invention
also provides
a method for potentiating T cell activation, which method comprises
administering an
effective amount of a multimerized neural cell adhesion molecule L1 (NCAM L1),
or a
functional derivative or fragment thereof, or a nucleic acid encoding said Ll
or functional
derivative or fragment thereof, or an agent that enhances production and/or
costimulatory
function of said L1 to a mammal, wherein T cell activation is desirable,
thereby
1 S potentiating T cell activation in said mammal.
Background Art
Current paradigms of T-cell activation are based on the premise that optimal
activation requires two signals; the first being provided by occupancy of the
T-cell receptor
(TCR) by MHC/antigen complex, the second being provided by one or more
costimulatory
ligands on the surface of the APC (1). An array of molecules on the surface of
the APC
can function as costimulatory ligands including members of the immunoglobulin
superfamily (IgSF) such as B7-1, B7-2 and ICAM-1 (1).
Previous studies identified Ll as a neuronal CAM that also belongs to the IgSF
(2).
To date, L1 function has almost exclusively been linked to neurological
processes,
including axonal guidance (3, 4). While such L1-mediated processes have
primarily been
attributed to homophilic L1-L1 ligation (5), this CAM can also interact with
multiple
heterophilic ligands including axonin 1/TAG l, chondroitin sulfate
proteoglycans, laminin
and certain integrins (6,7). L1 has also been shown to support cis-
interactions with the heat
stable antigen CD24 (~) and the tetraspan molecule CD9 (9).
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Despite its neuronal designation, Ll expression has recently been described on
cells
of both lymphoid and myelomonocytic origin (10, 11). Specifically, L1 can be
detected on
freshly isolated peripheral blood monocytes and on functionally mature
monocyte-derived
dendritic cells (DC) and on follicular DC in situ (11). Further constitutive
expression is
evident on a subset of B-cells and has been described on CD4+ T-cells (10,11).
Despite
these findings, little is known of the function of L1 in the immune system.
One recent
study has shown that Ll is important for the maintenance of lymph node
architecture (12).
Using a variety of experimental approaches, including one-way MLR and mitogen-
activation assays, we demonstrate here that Ll can function as a costimulatory
molecule in
T-cell activation. In this capacity, L1 contributes to the initiation of human
immune
responses in normal and disease processes including those involving the
nervous system.
Accordingly, it is an object of the present invention to provide methods for
modulating T cell activation using NCAM Ll as the modulating target. It is
another
objective of the present invention to provide combinations and combinatorial
methods foi
modulating T cell activation.
Disclosure of the Invention
This invention relates generally to the field of immunology or
neuroimmunology.
In one aspect, the invention provides a method for potentiating T cell
activation, which
method comprises administering an effective amount of a multimerized neural
cell
adhesion molecule Ll (NCAM L1), or a functional derivative or fragment
thereof, or a
nucleic acid encoding said L1 or functional derivative or fragment thereof, or
an agent that
enhances production and/or costimulatory function of said L1 to a mammal,
wherein T cell
activation is desirable, thereby potentiating T cell activation in said
mammal.
Any multimerized, e.g., dimerized, NCAM Ll, or a functional derivative or
fragment thereof, that can function as a stimulatory molecule in T cell
activation, and any
nucleic acids encoding such NCAM Ll, or functional derivative or fragment
thereof, can
be used in the present methods. Preferably, the NCAM L1, or a functional
derivative or
fragment thereof, is capable of L1-L1 homophilic interaction, e.g., mediating
a Ll-Ll
ligation between an antigen presentation cell (APC) and. a T cell. Also
preferably, the
NCAM L1, or a functional derivative or fragment thereof, supports an
interaction with an
integrin involved in T cell activation, e.g., supporting a traps or cis
interaction with the
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integrin cx5~31 or av~i3. Further preferably, the NCAM L1, or a functional
derivative or
fragment thereof, supports an interaction with a ligand involved in
costimulation, e.g.,
supporting a cis-type interaction with CD9 and/or CD24.
Any agents that enhances production and/or costimulatory function of NCAM Ll
can be used in the present methods. Preferably, the agents used therein
enhance Ll-Ll
homophilic interaction between two NCAM L1, or a functional derivative or
fragment
thereof, or interaction between a NCAM Ll, or a functional derivative or
fragment thereof,
and an integrin involved in T cell activation, or interaction between a NCAM
L1, or a
functional derivative or fragment thereof, and a ligand involved in
costimulation. One
exemplary agent is the anti-NCAM L1 monoclonal antibody 557.B6 (Appel et al.,
J.
Neurobiol., 28 3 :297-312 (1995)).
NCAM Ll, or a functional derivative or fragment thereof, from any mammalian
origins can be used. Preferably, when the mammal to be treated is a human, the
NCAM
Ll, or a functional derivative or fragment thereof, of human origin is used..
The present methods can be used to activate CD4+ T cells, CD8+ T cells or
both.
The present methods can be used to treat, either prophylactically or
therapeutically,
mammals with diseases or disorders associated with deficient T cell
activation. Examples
of such diseases or disorders include, but are not limited to, tumors, cancers
and infections.
Mammals, preferably humans, with tumors, cancers or infections are treated
with the
present methods.
In another aspect, the invention is directed to a combination, which
combination
comprises: a) an effective amount of multimerized NCAM Ll or a functional
derivative or
fragment thereof, or a nucleic acid encoding said L1 or functional derivative
or fragment
thereof, or an agent that enhances production and/or costimulatory function of
said Ll; and
b) an effective amount of another costimulatory molecule. Preferably, the
combination is
in the form of a pharmaceutical composition. Additionally, the invention is
directed to a
method for potentiating T cell activation, which method comprises
administering an
effective amount of multimerized NCAM Ll or a functional derivative or
fragment thereof,
or a nucleic acid encoding said Ll or functional derivative or fragment
thereof, or an agent
that enhances production and/or costimulatory function of said L1 and an
effective amount
of another costimulatory molecule to a mammal, wherein T cell activation is
desirable,
thereby potentiating T cell activation in said mammal. Any costimulatory
molecules can be
used in the above combinations and methods. Preferably, the costimulatory
molecules used
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are CD28, OX40, 4-1BB or ICOS. Also preferably, the costimulatory molecule is
derived
from an antigen presenting cell (APC), e.g., LFA-1, LFA-3, ICAM-1, ICAM-2,
ICAM-3,
CD 40 or B7.
In still another aspect, the invention also provides a method for reducing or
inhibiting T cell activation, which method comprises administering an
effective amount of
an antagonist of NCAM Ll to a mammal, wherein reduction or inhibition of T
cell
activation is desirable, thereby reducing or inhibiting T cell activation in
said mammal.
Any antagonists that reduce or inhibit production and/or costimulatory
function of
NCAM L1 can be used in the present methods. The antagonists can be NCAM L1
anti-
sense oligonucleotides, anti-NCAM L1 antibodies, especially monoclonal
antibodies such
as mAb SG3, soluble NCAM Ll, or derivatives or fragments thereof. The
antagonists can
reduce or inhibit Ll-Ll homophilic interaction, e.g., Ll-Ll ligation between
an antigen
presentation cell and a T cell. The antagonists can reduce or inhibit a L1-Ll
ligation
without simultaneously causing NCAM Ll clustering and signaling. The
antagonists can
reduce or inhibit NCAM L1's interaction with an integrin involved in T cell
activation, e.g.,
NCAM Ll's tans or cis interaction with the integrin a5~31 or av~33, or reduce
or inhibit
NCAM Ll's interaction with a ligand involved in costimulation, e.g., NCAM Ll's
interaction with CD9 and/or CD24. Preferably, the NCAM Ll antagonists used in
the
methods or combinations are protein, polypeptide ar peptide antagonists. Also
preferbaly,
the NCAM Ll antagonists used in the methods or combinations are small molecule
antagonists, e.g., ethanol (Bearer et al., J. Biol. Chem., 274 19 :13264-13270
(1999)).
The present methods can be used to reduce or inhibit activation of CD4+ T
cells,
CD8+ T cells or both.
The present methods can be used to treat, either prophylactically or
therapeutically,
mammals with diseases or disorders associated with undesirable T cell
activation.
Examples of such diseases or disorders include, but are not limited to,
autoimmunity, graft
rejection and neuroimmunological disorders. Mammals, preferably humans, with
autoimmunity, graft rejection and neuroimmunological disorders are treated
with the
present methods.
In yet another aspect, the invention is directed to a combination, which
combination
comprises: a) an effective amount of an antagonist of NCAM L1; and b) an
effective
amount of another costimulatory inhibitory molecule. Preferably, the
combination is in the
form of a pharmaceutical composition. Additionally, the invention is directed
to a method
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for reducing or inhibiting T cell activation, which method comprises
administering an
effective amount of an antagonist of NCAM L1 and an effective amount of
another
costimulatory inhibitory molecule to a mammal, wherein T cell reduction or
inhibition is
desirable, thereby reducing or inhibiting T cell activation in said mammal.
Any
costimulatory inhibitory molecules can be used in the above combinatioris~and
methods.
Preferably, the costimulatory inhibitory molecules used is T-lymphocyte-
associated antigen
4 (CTLA-4).
Brief Description of the Drawings
Figure 1. L1 is expressed by cord blood-derived DC and contributes to
allogenic
MLR. (A), CD34+-cells were enriched from normal cord blood and expanded for 21
days
prior to staining for Ll-expression. Enriched CD34+-cells were also stained
for Ll prior to
culture (Day 0). The cells were stained with mAb SG3 directly conjugated to
FITC. (B),
Cord blood derived DC (DC+) were cocultured with PBMC from a different donor
in an
one way MLR. Cells were cultured in the presence or absence of anti-Ll mAb SG3
or with
control antibody UPC10. CD34-negative cord blood cells cultured under
identical
conditions as the CD34+ enriched fraction were also tested as stimulators (non-
DC).
Cultures were pulsed with [3H]-thymidine during the last 18 hours of a three
day coculture.
Treatments were performed in triplicate. Error bars are ~1SE.
Figure 2. Anti-L1 antibody SG3 and soluble Ll inhibit autologous T-cell
responses
to mitogen. (A) PBMC were treated with PHA (1 Omg/ml) in the absence or
presence of
mAb SG3, or in the presence of control antibody UPC10. Further PBMC were
cultured in
the presence of soluble recombinant L1-ECD (sLl; 100mg/ml). (B) PBMC were
treated
with range of PHA concentrations in the absence or presence of mAb SG3, or in
the
presence of control antibody UPC10. Cultures were pulsed with [3H]-thymidine
during the
last 18 hours of a three day coculture. (B, inset) Some cells were treated
with PHA and
pulsed with [3H]-thymidine for 18 hours only. (C) Enriched CD4+ or CD8+ T-cell
subsets
were treated with PHA in the absence or presence of mAb SG3, or in the
presence of
control antibody UPC10. Cultures were pulsed with [3H]-thymidine during the
last 18
hours of a three day coculture. Treatments were performed in triplicate. Error
bars are
~1SE.
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Figure 3. Purified immobilized Ll potentiates T-cell proliferation in response
to
CD3 ligation. Wells of a 96-well plate were pretreated with anti-CD3 antibody
(OKT3:
25U/ml), with purified Ll-ectodomain (40mg/ml) or with a combination of both
Ll and the
mtibody. After washing the wells, PBMCs were added to the precoated wells or
to
untreated wells for 72 hours. PBMC were cultured in the absence or presence of
mAb SG3,
or in the presence of control antibody UPC10. Cultures were pulsed with [3H]-
thymidine
during the last 18 hours of a three day coculture. Treatments were performed
in triplicate.
Error bars are ~1SE.
Figure 4. Transfection and de novo expression of Ll enhances MLR. (A & B)
Irradiated wildtype (WT cells) or L1-transfected J558L myeloma cells (L1+
cells) were
cocultured with PBMC (A) or enriched CD4+ or CD8+ T-cell subsets (B) in an one
way
MLR. L1+ J558L myeloma cells were co-cultured in the presence or absence of
anti-Ll
mAb SG3 (80mg/ml) or in the presence of control mAb UPC10 (80mg/ml). (A,
inset)
Further co-cultures of Ll+ J558L myeloma cells and PBMC were incubated with
mAb SG3
at concentrations varying from 20-160mg/ml (inset). Cultures were pulsed with
[3H]=
thymidine during the last 18 hours of a three day coculture. Treatments were
performed in
triplicate. Error bars are ~1SE.
Figure 5. Inhibitory antibody SG3 blocks L1-Ll mediated adhesion by L1-
transfected myeloma cells. Wildtype (WT) or Ll-transfected (L1+) myeloma cells
were
allowed to adhere to immobilized recombinant Ll in the presence of absence of
control
mAb (CTPC10) or anti-L1 mAb (5G3). Adherent cells were counted per unit area
with a
40X high powered objective. Experimental treatments were performed in
triplicate with
four areas counted per well. Error bars represent + 1 SD.
Modes of Carr n~'n,~ Out the Invention
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the
same meaning as is commonly understood by one of ordinary skill in the art to
which this
invention belongs. All patents, applications, published applications and other
publications
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and sequences from GenBank and other data bases referred to herein are
incorporated by
reference in their entirety.
As used herein, "T cell activation" refers to cellular activation of resting T
cell
manifesting a variety of responses that include T cell proliferation, cytokine
secretion
and/or effector function. T cell activation may be induced by stimulation of
the T cell
receptor (TCR) with antigen/MHC complex. Alternatively, T cell activation may
be
induced by specified lectins, e.g., phytohemagglutinin, or monoclonal
antibody(ies) to
TCR.
As used herein, "costimulatory molecule" refers to molecules that modulate the
outcome of prior engagement of the TCR augmenting T cell activation events
including T-
cell proliferation and effector function. Signals provide by costimulatory
molecules are not
antigen specific nor MHC-restricted, and by themselves, i.e., in the absence
of TCR
engagement, axe unable to induce a significant response in T cells. Engagement
of the
TCR in the absence of costimulatory molecules result in no immune response or
hyporesponsiveness.
As used herein, "neural cell adhesion molecule Ll (NCAM Ll)" refers to a
neural
cell adhesion molecule that belongs to the IgSF superfamily and can function
as a
costimulatory molecule in T cell activation. NCAM L1 can exerts its
costimulatory
function through Ll-Ll homophilic interaction, e.g., mediating a Ll-Ll
ligation between
APCs and T cells or through interaction with an integrin involved in T cell
activation, e.g.,
the integrin a5~31 or av~33, or through interaction with a ligand involved in
costimulation,
e.g., CD9 and/or CD24. Preferably, NCAM L1 has 6 immunoglobulin like domains,
and
has 5 fibronectin type III like domains, and is a membrane-penetrating type
glycoprotein
expected to penetrate the membrane at a region having sufficient number, e.g.,
23,
hydrophobic amino acid residues starting with an amino acid with a small side
chain, e.g.,
glycine (EP 0,572,664 Al; and Moos et al., Nature, 334: 701-703 (1988)). It is
intended
that NCAM Ll includes those variants with conservative amino acid
substitutions that do
not substantially alter its costimulatory activity. Suitable conservative
substitutions of
amino acids are known to those of skill in this art and may be made generally
without
altering the biological activity of the resulting molecule. Those of skill in
this art recognize
that, in general, single amino acid substitutions in non-essential regions of
a polypeptide do
not substantially alter biological activity (see, ~, Watson et al. Molecular
Biology of tlae
Gene, 4th Edition, 1987, The BejacminlCummings Pub. co., p.224).
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As used herein: stringency of hybridization in determining percentage mismatch
is
as follows:
1) high stringency: 0.1 x SSPE, 0.1% SDS, 65°C
2) medium stringency: 0.2 x SSPE, 0.1% SDS, 50°C
3) low stringency: 1.0 x SSPE, 0.1% SDS, 50°C
It is understood that equivalent stringencies may be achieved using
alternative buffers, salts
and temperatures.
As used herein, a "functional derivative or fragment of NCAM Ll" refers to a
derivative or fragment of NCAM Ll that still substantially retains its
function as a
costimulatory molecule. Normally, the derivative or fragment retains at least
1%, 10%,
20%, 30%, 40%, 50% of its costimulatory activity. Preferably, the derivative
or fragment
retains at least 60%, 70%, 80%, 90%, 95%, 99% and 100% of its costimulatory
activity.
Functional derivative or fragment of NCAM Ll also encompasses peptide or
polypeptide
derivative or fragment of NCAM Ll that substantially retains its function as a
costimulatory molecule.
As used herein, an "agent that enhances production of NCAM Ll" refers to a
substance that increases transcription and or translation of a NCAM Ll gene,
or a
substance that increases post-translational modification andlor cellular
trafficking of a
NCAM L1 precursor, or a substance that prolongs half life of a NCAM Ll
protein.
As used herein, an "agent that enhances costimulatory function of NCAM L1"
refers to a substance that increases potency of NCAM Ll's costimulatory
activity, or a
substance that increases sensitivity of a NCAM L1's natural ligand in a
costimulatory
signally pathway, or a substance that decreases potency of a NCAM Ll's
antagonist.
As used herein, "integrins" refers to a family of cell membrane glycoproteins
that
are heterodimers composed of a- and ~3-chain subunits. They serve as
glycoprotein
receptors involved in cell-cell or cell-substrate adhesion, e.g., the
mediation of adhesion of
neutrophils to endothelial cells, or to extracellular matrix such as collagen.
As used herein, "neoplasm (neoplasia)" refers to abnormal new growth, and thus
means the same as tumoY, which may be benign or malignant. Unlike hyperplasia,
neoplastic proliferation persists even in the absence of the original
stimulus.
As used herein, "cancer" refers to a general term for diseases caused by any
type of
malignant tumor.
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As used herein, an "antagonist of NCAM L1 (or NCAM L1 antagonist)" refers to a
substance that decreases production and/or costimulatory function of NCAM L1.
Such an
antagonist can decrease production of NCAM L1 by decreasing transcription and
or
translation of a NCAM L1 gene, or by decreasing post-translational
modification and/or
cellular trafficking of a NCAM L1 precursor, or by shortening half life of a
NCAM L1
protein. Such an antagonist can decrease costimulatory function of NCAM Ll by
decreasing potency of NCAM Ll's costimulatory activity, or by decreasing
sensitivity of a
NCAM Ll's natural ligand in a costimulatory signally pathway, or by increasing
potency
of a NCAM Ll's antagonist. NCAM Ll antagonist can be any type of substances,
including protein, polypeptide, peptide, or small molecule antagonist. .
. As used herein, "antibody" includes antibody fragments, such as Fab
fragments,
which are composed of a light chain and the variable region of a heavy chain.
As used herein, a "combination" refers to any association between two or among
more items.
As used herein, a "composition" refers to a any mixture of two or more
products or
compounds. It may be a solution, a suspension, liquid, powder, a paste,
aqueous, non-
aqueous or any combination thereof.
As used herein, "antisense polynucleotides" refer to synthetic sequences of
nucleotide bases complementary to mRNA or the sense strand of double stranded
DNA.
Admixture of sense and antisense polynucleotides under appropriate conditions
leads to the
binding of the two molecules, or hybridization. When these polynucleotides
bind to
(hybridize with) mRNA, inhibition of protein synthesis (translation) occurs.
When these
polynucleotides bind to double stranded DNA, inhibition of RNA synthesis
(transcription) .
occurs. The resulting inhibition of translation and/or transcription leads to
an inhibition of
the synthesis of the protein encoded by the sense strand.
As used herein, an "NCAM Ll antisense oligonucleotide" refers to any oligomer
that prevents production or expression of NCAM L1 polypeptide. The size of
such an
oligomer can be any length that is effective for this purpose. In general, the
antisense
oligomer is prepared in accordance with the nucleotide sequence of a portion
of the
transcript of NCAM Ll that includes the translation initiation codon and
contains a
sufficient number of complementary nucleotides to block translation.
As used herein, an "autoimmunity" refers to specific humoral or cell-mediated
immune response to the body's own tissues.
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For clarity of disclosure, and not by way of limitation, the detailed
description of
the invention is divided into the subsections that follow.
B. Methods for potentiating T cell activation
In one aspect, the invention provides a method for potentiating T cell
activation,
which method comprises administering an effective amount of a multimerized
neural cell
adhesion molecule L1 (NCAM Ll), or a functional derivative or fragment
thereof, or a
nucleic acid encoding said Ll or functional derivative or fragment thereof, or
an agent that
enhances production and/or costimulatory function of said L1 to a mammal,
wherein T cell
activation is desirable, thereby potentiating T cell activation in said
mammal.
Any multimerized, e.g., dimerized, NCAM L1, or a functional derivative or
fragment thereof, that can function as a stimulatory molecule in T cell
activation, and any
nucleic acids encoding such NCAM Ll, or functional derivative or fragment
thereof; can
be used in the present methods.
For example, NCAM Ll proteins with the following GenBank accession numbers
can be used: T30532 (Fugu rubripes); T30581 (zebra fish); 536126 (rat); A43425
(chicken); 505479 (mouse); A41060 (human); NP 032504 (Mus musculus); NP 006605
(close homologue of Ll Sapiens); NP 000416 (Homo Sapiens); AAF22153 (Mus
musculus); CAB57301 (Mus musculus); P32004 (HTJMAN); Q05695 (RAT); P11627
(MOUSE); AAD28610 (Cercopithecus aethiops); CAB37831 (Homo sapiens); AACS 1746
(Homo sapiens); AAC15580 (Fugu rubripes); AAC14352 (Homo Sapiens); CAA96469
(Fugu rubripes); CA.A82564 (Homo Sapiens); CAA41576 (Homo Sapiens); 1411301A;
CAA42508 (Homo Sapiens); CAA41860 (Rattus norvegicus); AAA99159 (Carassius
auratus); CAA61491 (Danio rerio); CAA61490 (Danio rerio); AAA59476 (Homo
Sapiens);
AAA36353 (Homo Sapiens). In addition, any proteins derived from, or are
portion of, the
above NCAM L1 proteins that still substantially retain their costimulatory
activities can be
used. Preferably, such NCAM L1 derivatives or fragments can be recognized by
antibodies
that specifically recognize the NCAM L1 proteins from which the derivatives or
fragments
originate.
Similarly, nucleic acids encoding NCAM L1 proteins with the following GenBank
accession numbers can be used: AC005775 (Homo Sapiens); AC 004690 (Homo
Sapiens);
M28231 (Drosophila melanogaster neuroglian precursor); AH006326 (Drosophila
melanogaster neuroglian (nrg), alternative splice products); AF050085
(Drosophila
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melanogaster neuroglian (nrg) gene; AF172277 (Homo sapiens); AF133093 (Mus
musculus); AJ239325 (Homo Sapiens); AL021940 (Homo sapiens); AF129167
(Chlorocebus aethiops); AJ011930 (Homo Sapiens); U52112 (Homo Sapiens); M97161
(Rattus norvegicus); AC005626 (Homo Sapiens); AF026198 (Fugu rubripes); M77640
(Homo Sapiens); U55211 (Carassius auratus); M74387 (Human). W addition, any
nucleic
acids derived from, or are portion of, the above nucleic acids encoding NCAM
Ll that still
substantially retain their costimulatory activities can be used. Preferably,
such NCAM Ll
nucleic acid derivatives or fragments can hybridize under low, middle or high
stringency
with the NCAM Ll nucleic acids fromwhich the derivatives or fragments
originate.
Preferably, the NCAM Ll, or a functional derivative or fragment thereof, is
capable
of Ll-L1 homophilic interaction, e.g., mediating a L1-Ll ligation between an
antigen
presentation cell (APC) and a T cell. Also preferably, the NCAM L1, or a
functional
derivative or fragment thereof, supports an interaction with an integrin
involved in T cell
activation, e.g., supporting a traps or cis interaction with the integrin
a5,~1 (Ruppert et al.,
J. Cell Biol., 131:1881-1891 (1995)), or integrin x5(33 (Sturmhofel et al., J.
Immunol.,
154 5 :2104-11 (1995); and Poul et al., Mol. Immunol., 32 2 :101-16 (1995)),
or CDl lc, a
,62 integrin (Meunier, et al., J. Ihvest. Dermatol., 103 6 :775-9 (1994)), or
VLA integrin
family (Dang, et al., J. Exp. Med., 172 2 :649-52 (1990)). Further preferably,
the NCAM
Ll, or a functional derivative or fragment thereof, supports an interaction
with a ligand
involved in costimulation, e.g., supporting a cis-type interaction with CD9
and/or CD24
(Liu et al., J. Exp. Med., 175::437-445 (1992); and Lagaudriere-Gesbert et
al., Cell.
Immuhol., 182:105-112 (1997)).
The NCAM L1, or functional derivative or fragment thereof, or the nucleic acid
encoding the NCAM L1, or functional derivative or fragment thereof, can be
administered
to the mammal by any methods know in the art. For example, the NCAM L1, or
functional
derivative or fragment thereof, or the nucleic acid encoding the NCAM Ll, or
functional
derivative or fragment thereof, can be administered directly to the mammal.
Alternatively,
the NCAM Ll, or functional derivative or fragment thereof, or the nucleic acid
encoding
the NCAM Ll, or functional derivative or fragment thereof, can be delivered
into antigen
presenting cells, e.g., macrophages and dendritic cells, and the antigen
presenting cells
containing the NCAM L1 or the nucleic acid are then administered to the
mammal.
Any agents that enhances production and/or costimulatory function of NCAM Ll
can be used in the present methods. Preferably, the agents used therein
enhance L1-Ll
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homophilic interaction between two NCAM LI, or a functional derivative or
fragment
thereof, or interaction between a NCAM L1, or a functional derivative or
fragment thereof,
and an integrin involved in T cell activation, or interaction between a NCAM
L1, or a
functional derivative or fragment thereof, and a ligand involved in
costimulation.
NCAM Ll~ or a functional derivative or fragment thereof, from any mammalian
origins can be used. Preferably, when the mammal to be treated is a human, the
NCAM
L1, or a functional derivative or fragment thereof, of human origin is used..
The present methods can be used to activate CD4+ T cells, CD~+ T cells or
both.
The present methods can be used to treat, either prophylactically or
therapeutically,
mammals with diseases or disorders associated with deficient T cell
activation. Examples
of such diseases or disorders include, but are not limited to, tumors, cancers
or infections.
Examples of tumors or cancers that can be treated with the present methods
include breast
cancer, Burkitt lymphoma, colon cancer, small cell lung carcinoma, melanoma,
multiple
endocrine neoplasia (MEN), neurofibromatosis, p53-associated tumor, pancreatic
carcinoma, prostate cancer, Ras-associated tumor, retinoblastoma and Von-
Hippel Lindau
disease (VHL). Preferably, tumors or cancers that originate from immune system
and/or
nervous system are treated.
Any mammals, such as, mice, rats, rabbits, cats; dogs, pigs, cows, ox, sheep,
goats,
horses, monkeys and other non-human primates, with tumors, cancers or
infections can be
treated with the present methods. Preferably, humans with tumors or cancers
are treated
with the present methods.
C. Methods for inhibiting T cell activation
In another aspect, the invention is directed to a method for reducing or
inhibiting T
cell activation, which method comprises administering an effective amount of
an antagonist
of NCAM LI to a mammal, wherein T cell reduction or inhibition is desirable,
thereby
reducing or inhibiting T cell activation in said mammal.
Any antagonists that reduce or inhibit production andlor costimulatory
function of
NCAM Ll can be used in the present methods. Preferably, the antagonists used
therein are
NCAM Ll anti-sense oligonucleotides, anti-NCAM Ll antibodies, especially
monoclonal
antibodies such as mAb SG3 (Balaian et al., Eur. J. Immunol., 30 3 :93g-43
(2000)),
soluble NCAM Ll, or derivatives or fragments thereof. Also preferably, the
antagonists of
NCAM L1 used reduce or inhibit L1-L1 homophilic interaction, e.g., L1-Ll
ligation
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between an antigen presentation cell and a T cell. More preferably, the
antagonists reduce
or inhibit a Ll-Ll ligation without simultaneously causing NCAM L1 clustering
and
signaling. Still preferably, the antagonists of NCAM Ll used reduce or inhibit
NCAM
Ll's interaction with an integrin involved in T cell activation, e.g., NCAM
Ll's traps or cis
interaction with the integrin x5(31, av(33, CD 11 c, a X32 integrin or VLA
integrin family, or
reduce or inhibit NCAM Ll's interaction with a ligand involved in
costimulation, e.g.,
NCAM L1's interaction with CD9 and/or CD24.
In a preferred embodiment, the NCAM Ll antagonists used in the methods or
combinations are protein, polypeptide or peptide antagonists. In another
preferred
embodiment, the NCAM Ll antagonists used in the methods or combinations are
small
molecule antagonists, e.g., ethanol (Bearer et al., J. Biol. Chem., 274 19
:13264-13270
( 1999)).
The present methods can be used to reduce or inhibit activation of CD4~ T
cells,
CD8+ T cells or both.
The present methods can be used to treat, either prophylactically or
therapeutically,
mammals with diseases or disorders associated with undesirable T cell
activation.
Examples of such diseases or disorders include, but are not limited to,
autoimmunity, graft
rejection and neuroimmunological disorders. Mammals, preferably humans, with
autoimmunity, graft rejection and neuroimmunological disorders are treated
with the
present methods.
D. Combinations and combinatorial treatments
In still another aspect, the invention is directed to a combination, which
combination comprises: a) an effective amount of multimerized NCAM Ll or a
functional
derivative or fragment thereof, or a nucleic acid encoding said L1 or
functional derivative
or fragment thereof, or an agent that enhances production andlor costimulatory
function of
said L1; and b) an effective amount of another costimulatory molecule, or an
immunostimulant such as an agonist of costimulatory molecules, or certain
cytokines, e.g.,
IL-2. Preferably, the combination is in the form of a pharmaceutical
composition.
Additionally, the invention is directed to a method for potentiating T cell
activation, which
method comprises administering an effective amount of multimerized NCAM Ll or
a
functional derivative or fragment thereof, or a nucleic acid encoding said Ll
or functional
derivative or fragment thereof, or an agent that enhances production andlor
costimulatory
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WO 02/28440 PCT/USO1/30864
function of said Ll and an effective amount of another costimulatory molecule
or an
immunostimulant to a mammal, wherein T cell activation is desirable, thereby
potentiating
T cell activation in said mammal. Any costimulatory molecules can be used in
the above
combinations and methods. Preferably, the costimulatory molecules used are
CD28,
0X40, 4-1BB or ICOS.
CD28 is the primary positive T cell costimulatory molecule, as defined by the
ability to enhance T cell activation in the presence of TCR stimulation that
is insufficient
for T cell proliferation (see generally Chambers and Allison, Curr. Opin.
Cell. Biol.,
11 2 :203-10 (1999)). CD28 is an immunoglobulin supergene family glycoprotein
that is
expressed as homodimers on T cells. It binds to ligands B7.1 and B7.2 via the
MYPPPY
(in the single letter code for amino acids) motif in the immunoglobulin
domain. The
cytoplasmic tail of CD28 possesses tyrosine-containing motifs postulated to be
involved in
signal transduction and protein trafficking. In a specific embodiment, CD28
protein with
the following GenBank accession numbers can be used in the combination and
combinatorial treatment method: NP-006130 (Homo Sapiens); B45895 (human);
I49584
(mouse); I46689 (rabbit); 524413 (rat); A43523 (mouse); RWHU28 (human);
AAF45150
(Mus musculus); BAA92349 (Felis catus); AAF36501 (Marmota monax); NP 037253
(Rattus norvegicus); AAF33794 (Homo sapiens); AAF33793 (Homo sapiens);
AAF33792
(Homo sapiens); BAA08641 (Oryctolagus cuniculus); ~ 031668 (Mus musculus)
NP-008820 (Homo Sapiens); NP-005182 (Homo Sapiens); Q28071 (Bovin); P42069
(rabbit); P31042 (rat); P31041 (mouse); P31043 (chick); P10747 (human) AD04379
(Ovis
arie gi); AAB53574 ( Felis catus); CAA63707 (Bos taurus); CAA39003 (Rattus
norvegicus); AAA51945 (Homo Sapiens); AAA51944 (Homo sapiens); AAA37395~(Mus
musculus); AAA37396 (Mus musculus). Similarly, nucleic acids encoding CD28
with the
following GenBank accession numbers can be used in the combination and
combinatorial
treatment method: AB025316 (Felis catus); AF130427 (Marmota monax); AF222343
(Homo sapiens); AF222342 (Homo sapiens); AF222341 (Homo sapiens); D49841
(rabbit);
AF092739 (ovis aries);~A1528690 (mouse); AI386096 (human); AI327367 (mouse);
AI324382 (mouse); AII52205 (mouse); AA940559 (mouse); U57754 (Felis catus);
AA17418 (human); AA163825 (mouse); J02988 (human); M34563 (mouse).
In another specific embodiment, 0X40 protein with the following GenBank
accession numbers can be used in the combination and combinatorial treatment
method:
137552 (0X40 homolog-human); JE0351 (rat); 148700 (mouse); 548290 (mouse);
512783
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(rat); 1DOAL 1DOAK (Chain L, Human); 1DOAJ (Chain J, Human); 1DOAI (Chain I,
Human); 1DOAH (Chain H, Human); 1DOAG (Chain G, Human); 1DOAF (Chain F,
Human); 1DOAE Chain E, Human); 1DOAD (Chain D, Human); 1DOAC (Chain C,
Human); 1DOAB (Chain B, Human); 1DOAA (Chain A, Human); NP 003318 (Homo
sapiens); CAA18438 (Homo Sapiens); 002765 (rabbit); P47741 (MOUSE); P43488
(MOUSE); P15725 (RAT); P23510 (HUMAN); AAC67236 (Rattus norvegicus);
BAA20060 (Oryctolagus cuniculus); BAA 20059 (Oryctolagus cuniculus); AAB33944
(human); CAA53576 (Homo sapiens); CAA79772 (Mus musculus); CAA59476 (Mus
musculus); AAA21871 (Mus musculus). Similarly, nucleic acids encoding OX40
with the
following GenBank accession numbers can be used in the combination and
combinatorial
treatment method: AL022310 (human); AF037067 (Rattus norvegicus); AB003912 .
(rabbit); U12763 (Mus musculus).
In still another specific embodiment, 4-1BB protein with the following GenBank
accession numbers can be used in the combination and combinatorial treatment
method:
138427 (human); 138426 (human); 153384 (mouse); B32393 (mouse); P41273
(human);
P41274 (mouse); Q07011 (human); P20334 (mouse); AAA93113 (mus musculus);
AAA53134 (Homo Sapiens); AAA53133 (Homo sapiens); AAA40167 (Mus musculus);
AAA39435 (Mus musculus). Similarly, nucleic acids encoding 4-1BB with the
following
GenBank accession numbers can be used in the combination and combinatorial
treatment
method: AI664286; AII57872; AA109726; AA389045; AA155147; AA087107; W62906;
U02567 (Mus musculus); U03398 (human); U03397 (human); J04492 (mouse); L15435
(Mus musculus).
Recently, a CD28/CTLA-4 homologue called ICOS has been cloned from activated
human T cells. ICOS has an structure similar to CD28 and CTLA-4 but does not
have a
conserved MYPPY motif, suggesting that it binds to unique ligand(s). Antibody
cross-
linking of ICOS enhances anti CD3-mediated T cell proliferation and cytokine
production,
although, unlike CD28, it does not enhance IL-2 production. ICOS protein and
nucleic
acid encoding ICOS protein with the following GenBank accession numbers can be
used in
the combination and combinatorial treatment method: 578540 (human) and
AJ250559
(Mus musculus).
Also preferably, the costimulatory molecule is derived from an antigen
presenting
cell (APC), e.g., LFA-1, LFA-3, ICAM-1, ICAM-2, ICAM-3, CD 40 or B7.
CA 02423436 2003-03-24
WO 02/28440 PCT/USO1/30864
In yet another aspect, the invention is directed to a combination, which
combination
comprises: a) an effective amount of an antagonist ofNCAM Ll; and b) an
effective
amount of another costimulatory inhibitory molecule. Preferably, the
combination is in the
form of a pharmaceutical composition. Additionally, the invention is directed
to a method
for reducing or inhibiting T cell activation, which method comprises
administering an
effective amount of an antagonist of NCAM Ll and an effective amount of
another
costimulatory inhibitory molecule to a mammal, wherein T cell reduction or
inhibition is
desirable, thereby reducing or inhibiting T cell activation in said mammal.
Any costimulatory inhibitory molecules can be used in the above combinations
and
methods. For example, the costimulatory inhibitory molecules can be
antagonists of
costimulatory molecules including the costimulatory molecules described above
such as
CD28, OX40, 4-1BB or ICOS and the costimulatory molecule is derived from an
antigen
presenting cell (APC), e.g., .LFA-1, LFA-3, ICAM-l, ICAM-2, ICAM-3, CD 40 or
B7.
In another example, the costimulatory inhibitory molecules used is T-
lymphocyte-
associated antigen 4 (CTLA-4) (Chambers and Allison, Curr. Opih. Cell. Biol.,
11 2 :203-
10 (1999)). CTLA-4 is an immunoglobulin supergene family glycoprotein that is
expressed as homodimers on T cells. It binds to ligands B7.1 and B7.2 via the
MYPPPY
(in the single letter code for amino acids) motif in the immunoglobulin
domain. CTLA-4
has a 10-fold higher affinity and a 100-fold higher avidity for B7 ligands
compared to
CD28 and exhibits distinct binding kinetics. The cytoplasmic tail of CTLA-4
possess
tyrosine-containing motifs postulated to be involved in signal transduction
and protein
trafficking. In another specific embodiment, CTLA-4 protein with the following
GenBank
accession numbers can be used in the combination and combinatorial treatment
method:
I46696 (rabbit); BAA08644 (oryctolagus cuniculus); P42081 (human); P42072;
P16410;
P09793; P33681; AAD50988 (Felis catus); AAD00698; AAD00697; (Rattus
norvegicus);
AD00696; (Mus musculus); IAHl; 2207257A; 1309302A; CAA63708 (Bos taurus);
CAA29191 (Mus musculus); AAA86473 (Homo sapiens). Similarly, nucleic acids
encoding CTLA-4 with the following GenBank accession numbers can be used in
the
combination and combinatorial treatment method: AF130428 (Marmota monax);
D49844
(rabbit); AF143204 (cams familiaris breed beagle); AF1701725 (Felis catus);
AF092740
(Ovis aries); AF153202 (Felix catus); U90271 (Rattus norvegicus); U37121
(Rattus
norvegicus); L15006 (Homo sapiens); U17722 (Human).
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WO 02/28440 PCT/USO1/30864
The formulation, dosage and route of administration of the above-described
compositions, combinations, preferably in the form of pharmaceutical
compositions, can be
determined according to the methods known in the art (see e.g., Remington: The
Science
and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company,
April
1997; Therapeutic Peptides and Proteins: Formulation, Processing, arzd
Delivery Systems,
Banga, 1999; and Pharmaceutical Formulation Development of Peptides and
Proteins,
Hovgaard and Frkjr (Ed.), Taylor & Francis, Inc., 2000; Medical Applications
of
Liposomes, Lasic and Papahadjopoulos (Ed.), Elsevier Science, 1998; Textbook
of Gene
Therapy, Jain, Hogrefe & Huber Publishers, 1998; Adezzoviruses: Basic Biology
to Gene
Therapy, Vol. 15, Seth, Landes Bioscience, 1999; Biopharmaceutical Drug Design
and
Development, Wu-Pong and Rojanasakul (Ed.), Humana Press, 1999; Therapeutic
Angiogenesis: From Basic Science to the Clinic, Vol. 28, Dole et al. (Ed.),
Springer-Verlag
New York, 1999). The compositions, combinations or pharmaceutical compositions
can be
formulated for oral, rectal, topical, inhalational, buccal (e.g., sublingual),
paxenteral (e.g.,
subcutaneous, intramuscular, intradermal, or intravenous), transdermal
administration or
any other suitable route of administration. The most suitable route in any
given case will
depend on the nature and severity of the condition being treated and on the
nature of the
particular composition, combination or pharmaceutical composition which is
being used.
The following example is included for illustrative purposes only and is not
intended
to limit the scope of the invention.
E. Examples
Ll-li~ation is required for optimal allo-stimulation
DCs are characterized as the most proficient APC in the immune system and are
recognized to be the principal stimulators of primary MLR. A population of
'stimulatory'
DC were produced from enriched CD34+ stem cells (>76% purity) using a
combination of
GM-CSF, SCF, IL-3, TNF-a, IL-4. After 14-21 days these cells had acquired the
expected
DC morphology and phenotype (CIDIa+, CD80+, CD86+, CD3-, CD14-, CD19-, CD56-)
(data not shown). Importantly, acquisition of this DC phenotype was also
marked by the
induction of L1 expression (Fig. 1A). Expression of Ll on these DC is in
accord with our
previous report describing Ll expression on precursor and monocyte-derived DC
(11). In
order to establish an allogeneic MLR the Ll+ DC were cocultured with PBMC from
a
different donor. The contribution of Ll to this allogeneic MLR was determined
by the
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WO 02/28440 PCT/USO1/30864
inclusion of a mAb specific for L1 (mAb SG3; 11). Importantly, this antibody
significantly
reduced the allogeneic PBL response while an isotype-matched control antibody
(UPC.10)
had no significant effect (Fig. 1B). Using PBMC from a number of different
donors we
observed levels of inhibition between 25-40% (not shown).
L1-li atg ion potentiates T-cell responses to PHA and to CD3
To further confirm costimulatory function in an autologous system we
determined
whether the anti Ll antibody SG3 would also reduce proliferative responses to
the T-cell
mitogen PHA. Importantly, blockade of Ll by this mAb significantly reduced T-
cell
proliferation within the PBMC fraction (Fig. 2A & B) and reduced the
proliferation of
enriched T-cell subsets, in particular CD4+ cells (Fig. 2C). Inhibtion by
antibody SG3 was
observed over a range of PHA concentrations (Fig. 2B) and as early as 18 hours
after PHA
stimulation (Fig. 2B inset).
Since we have previously demonstrated that mAb SG3 will recognize monocytes in
freshly isolated PBMC (11), it is likely that this antibody can inhibit
mitogen-driven T=cell
proliferation by blocking Ll expressed by these accessory cells. In this
regard, either the
removal of accessory cells or the blockade of costimulatory molecules
expressed by these
cells is known to abrogate or reduce T-cell responses to PHA. The importance
of accessory
cell function was confirmed in this study by the lower mitogenic responses of
the isolated
CD4+ and CD8+ T-cell subsets which were enriched to approximately 95% purity
(Fig. 2).
Finally, it is important to note that purified recombinant Ll (Ll-ECD) was
also found to
reduce T-cell proliferation when offered as a soluble inhibitor (sLl : Fig.
2A).
To further demonstrate costimulatory function it was determined whether
purified
immobilized Ll could potentiate polyclonal T-cell proliferation in response to
ligation of
the CD3 receptor (mAb OKT3). PBMC were added to wells precoated with mAb OKT3
alone or in combination with purified Ll ectodomain (L1-ECD). While wells
coated with
Ll alone failed to induce a significant response, the Ll did markedly enhance
responses to
the anti-CD3 mAb (Fig. 3). The specificity of this synergistic response was
confirmed by
inhibition with mAb SG3 (Fig. 3). It is important to note that mAb SG3 will
block
homophilic Ll-mediated adhesion to purified L1 ECD but will not block integrin-
dependent adhesion to the same L1-ECD preparation (not shown). This data
confirms that
Ll can function as a potent costimulatory molecule, and indicates that
homophilic Ll-L1
binding rather than direct L1-integrin binding is the stimulatory mechanism.
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Ll-transfection of myeloma cells promotes adhesion via a hemophilic mechanism
and potentiates MLR
To directly demonstrate costimulatory function it should be pbssible to show
that
transfection and de novo expression of L1 will enhance the ability of the
transfected cell to
promote T-cell activation. We therefore compared the response of human PBMC or
enriched T-cells to either wildtype or Ll transfected marine myeloma cells
(J558L) in an in
a one-way xenogeneic MLR. Importantly, irradiated myeloma cells manipulated
to.express
high levels of human Ll (13), were found to be significantly more efficient at
inducing
PBMC proliferation than their wildtype counterparts (Fig. 4A). The specific
contribution
of Ll to this enhanced MLR was confirmed by inhibition with mAb SG3 (Fig. 4A).
Both
CD4+ and CD8+ T-cells were found to respond differentially to the Ll
transfected
myeloma cells, however, the response of the CD4+ cells was found to be
superior (Fig.
4B). The ability of the Ll-transfected myeloma cells to induce T-cell
proliferation was lost
if these cells were cocultured with mononuclear cells derived from BALB/c mice
(not
shown). Since the J558L myelorila line was originally derived from a BALB/c
mouse this
would suggest that the ability of L1 to stimulate the proliferation of human T-
cells is
dependent upon a simultaneous recognition of, and response to, marine
xenoantigens. In
this regard, we did observe some T-cell proliferation in response to the
wildtype Ll-
negative J558L cells (fig. 4B).
The introduction of L1 onto the surface of the myeloma cells was also found to
facilitate adhesion to purified recombinant Ll (Ll-ECD) via a hemophilic
mechanism (Fig.
5). These data suggest that the enhanced MLR observed with the L1-
transfectants is due to
a de novo capacity for L1-L1 interaction and adhesion. It is important to
note, that the Ll-
mediated adhesion observed was completely abrogated by mAb SG3 (Fig. 5)
suggesting
that this antibody can inhibit T-cell activation by virtue of its ability to
prevent L1-Ll
hemophilic interaction.
It should be noted that inhibition of T-cell activation may depend upon the
use of
antagonists that can block Ll-ligation without simultaneously causing Ll
clustering and
signalling. Supporting this concept we did not observe any significant
inhibition of T-cell
activation using an anti-L1 polyclonal antibody (data not shown). In this
regard, it has
been documented that polyclonal antibodies to Ll (unlike most mAbs and
isolated soluble
Ll) can result in the activation of L1 dependent signalling pathways resulting
the
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WO 02/28440 PCT/USO1/30864
significant increases in intracellular cAMP levels (14). Finally, it was also
observed that
the inhibitory activity of SG3 antibody preparations was critically dependent
upon the
removal of antibody aggregates by ultracentrifugation and that activity was
lost both on
freezing and concentration.
Concluding Remarks
Despite being described as a neural CAM we have recently documented Ll
expression by 'professional' APC of both myelomonocytic and lymphoid origin,
including
B-cells, activated monocytes, monocyte derived DC, and follicular DC (11). L1
expression
IO on monocyte-derived DC was induced after treatment with LPS (I 1) which is
known to
promote functional maturation or the acquisition of optimal costimulatory
capacity. Based
on these findings, and those present in this study, we propose that L1
expressed by such
'professional' APC can function as a costimulatory molecule in T-cell
activation. A recent
report documenting the expression of Ll by isolated peripheral blood T-cells
(10) suggests
a possible costimulatory mechanism based on homophilic L1-Ll ligation between
the APC
and T-cell. Adding support to this mechanism, we show that T-cell activation
is inhibited
by an anti-Ll mAb that also effectively prevents Ll-L1 ligation.
Further detailed studies are required to define how L1-mediated signalling
potentiates T-cell costimulation and to determine how Ll ranks along side
other
costimulatory molecules. A comparison with previous studies suggests that Ll
is less
important for T-cell co-stimulation than, for example, members of the B7
family. Thus
inhibition of B7.1B7.2 has been shown to reduce T cell proliferation to PHA
and to
allogeneic DC by up to 75 and 95% respectively (15, 16, 17). However, Ll-
mediated
costimulation may be compared with other well documented co-stimulatory
molecules such.'
as CD58. Thus, blockade of CD2;CD58 binding has been shown to inhibit T-cell
proliferation to PHA and to allogeneic DC by 30-35 and 45-50% respectively
(16, 17).
While our findings indicate that homophilic L1-LI ligation is required for T-
cell
costimulation, it is important to note that Ll can undergo multiple cis and
trans interactions
with other heterophilic ligands (6). For example, Ll has recently been shown
to support a
trans interaction with the integrin aSbl (18); an integrin which has also been
implicated in
T-cell activation (19). It is conceivable that Ll-integrin binding can
contribute to
costimulation after an initial T-cell activation event which is required for
subsequent
integrin activation and binding. In addition, it is notable that Ll can
undergo cis-type
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WO 02/28440 PCT/USO1/30864
interactions with both CD9 and CD24 both of which are known to play an
important role
in costimulation (20, 21).
Several important ramifications arise from the findings presented. Like other
costimulatory molecules, L1 may contribute to the development of autoimmunity,
graft
rejection, and anti-tumor responses and in this context may prove to be a
useful and novel
target for immunotherapeutic intervention. The finding that soluble Ll can
inhibit T-cell
activation may prove significant given reports that aggressive neuroectodermal
tumors can
secrete laxge amounts of L1 (13, 22). Finally, high levels of Ll-expression on
post mitotic
neurons and Schwann cells (2,6) suggest that this CAM may function as an
important
intermediary between nervous and immune system, particularly in the
development of
neuroimmunological disorders.
Materials and Methods
Reagents and Cell Lines
Anti-human Ll mAb SG3 was generated in our laboratory (19). Purified
recombinant Ll consisting of the entire extracellular domain of human Ll (Ll-
ECD) was
kindly provided by Dr William Stallcup (The Burnham Institute, La Jolla, CA).
J558L.
myeloma cells stably transfected with the full length human cDNA encoding for
human Ll
(J558L-L1; 21) were kindly provided by Dr Vance Lemmon (ease-Western Reserve
University, OIT).
Generation of DC and Enrichment of CD4 and CD8 T-lymphocytes
CD34+ cells were purified from normal cord blood using M-450 Dynabeads coated
with an anti-CD34 mAb according to the manufacturers instructions. CD34+-
enriched
(>76%) or CD34 negative cell populations were then cultured in the presence of
granulocyte-macrophage colony-stimulating factor (l0ng/ml), human stem cell
factor (40
ng/ml), human interleukin-3 (10 ng/ml), human tumor necrosis factor-a (100
U/mL) and
human interleukin-4 (400U/mL). After expansion for 7-21 days the levels of Ll
expression on the cells was determined using anti-Ll mAb SG3 directly
conjugated to
fluorescein isothiocyanate (FITC).
CD4+ and CD8+ cells were isolated from PBMC using M-450 Dynabeads coated
with anti-CD4 or anti-CD8 mAbs. Isolation was according the manufacturers
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CA 02423436 2003-03-24
WO 02/28440 PCT/USO1/30864
recommendations (Dynal, Fort Lee, N.J.). This method does not induce T-cell
activation
and resulted in approximately 95% purity.
MLR and Mito~en Assays
For mitogen assays, PBMC, or enriched CD4+, or CD8+ cells were cultured in 96-
well round bottom plates (1 x 105 cells/well), with or without PHA (Sigma; 20
mg/ml).
For MLR assays, PBMCs (1 x 105 cells/well) or enriched CD4+, or CD8+ cells
were co-
cultured with irradiated wildtype or Ll-transfected J558L cells at 1 x 104
cells/well or with
irradiated cord blood derived dendritic cells (1 x 104 cells/well). Mitogen
treated cells and
cocultures were maintained for 3 days and the cultures pulsed with [3H]-
thymidine-(1
mCi/well) during the last 18 hours of the three-day culture. The contribution
of Ll to both
mitogen and MLR assays was assessed by the incorporation of anti-Ll mAb SG3
(80mg/ml). Where appropriate an IgG2a isotype-matched control antibody
(ITPC10;
80mg/ml) was also added.
Co-stimulation of CD3-mediated T-cell activation
Wells of a 96-well plate were pretreated with anti-CD3 antibody OKT3 (25
U/ml),
with purified Ll-ectodomain (40mg/ml) or with a combination of both Ll and the
antibody.
After washing the wells, PBMCs were added to the precoated wells or to
untreated wells
for 72 hours. PBMC were added at 1 x 105 cells/well in the absence or presence
of mAb
SG3, or in the presence of control antibody UPC 10. Cultures were pulsed with
[3H]-
thymidine during the last 18 hours of a three day coculture. Treatments were
performed in
triplicate. Error bars are ~1 SE.
Adhesion Assay
Purified Ll-ECD fusion protein in PBS (30mg/ml) was coated onto the bottom of
96-well Titertek plates essentially as described (13). The wells were then
blocked with 5%
BSA and wildtype or Ll transfected J558L myeloma cells added at 1 X 105/well
and
allowed to adhere for 60 minutes at 37°C. Cells were added in HBSS
supplemented with
lOmM Hepes, BSA (0.5%) and CaCl2 (O.SmM) (pH 7.4). For inhibition studies, the
cells
were pretreated with anti-L1 mAb SG3 or isotype matched control mAb UPC10 at
80
mg/ml prior to the addition of both cells and inhibitors to pre-treated wells.
Non-adherent
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cells removed under a constant vacuum and remaining adherent cells enumerated
using a
40X objective as described (13).
References
1. Croft, M. and Dubey, C., Accessory molecule and costimulation requirements
for CD4 T
cell responses. Crit. Rev. Iminunol. 1997. 17: 89-118.
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Since modifications will be apparent to those of skill in this art, it is
intended that
this invention be limited only by the scope of the appended claims.
