U.S. patent application number 12/451011 was filed with the patent office on 2010-06-03 for method for modularting activity of t lymphocytes.
Invention is credited to Thierry Boon-Falleur, Nathalie Demotte, Peirre Van Der Druggen.
Application Number | 20100136597 12/451011 |
Document ID | / |
Family ID | 39925960 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100136597 |
Kind Code |
A1 |
Demotte; Nathalie ; et
al. |
June 3, 2010 |
METHOD FOR MODULARTING ACTIVITY OF T LYMPHOCYTES
Abstract
The invention relates to methods and compositions which modulate
T lymphocyte activity. It has been found that two, T lymphocyte
receptors, especially TCR and CD8, are present at a distance from
each other on T lymphocyte surfaces. Via use of modulators which
change the distance between these receptors, the activity of the T
lymphocyte is modulated.
Inventors: |
Demotte; Nathalie; (Chastre,
BE) ; Van Der Druggen; Peirre; (Chaumont-Gristoux,
BE) ; Boon-Falleur; Thierry; (Bruxelles, BE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
39925960 |
Appl. No.: |
12/451011 |
Filed: |
March 3, 2008 |
PCT Filed: |
March 3, 2008 |
PCT NO: |
PCT/US08/02774 |
371 Date: |
February 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60926265 |
Apr 26, 2007 |
|
|
|
Current U.S.
Class: |
435/29 ;
435/235.1; 435/375 |
Current CPC
Class: |
C12N 5/0636 20130101;
A61K 31/7016 20130101; A61K 45/06 20130101; C12N 2501/73 20130101;
C12N 2501/59 20130101; A61K 31/4745 20130101; A61K 2039/5158
20130101; C07K 14/70517 20130101; A61K 31/4745 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; C12N 2500/22 20130101;
C07K 14/7051 20130101; G01N 33/505 20130101; A61K 31/7016 20130101;
C12N 2500/34 20130101 |
Class at
Publication: |
435/29 ; 435/375;
435/235.1 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12N 5/00 20060101 C12N005/00; C12N 7/00 20060101
C12N007/00 |
Claims
1. A method for modulating activity of a T lymphocyte, comprising
contacting said T lymphocyte with a substance that changes distance
between a T lymphocyte receptor and a T lymphocyte co-receptor to a
point sufficient to modulate activity of said T lymphocyte.
2. The method of claim 1, wherein said T lymphocyte co-receptor is
a CD8 molecule.
3. The method of claim 1, wherein said T lymphocyte receptor is a T
cell receptor molecule.
4. The method of claim 1, wherein said substance reduces distance
between a T lymphocyte receptor and a T lymphocyte co-receptor and
increases activity of said T lymphocyte.
5. The method of claim 2, wherein said substance is a zinc
chelator.
6. The method of claim 4, wherein said zinc chelator is
o-phenantholin.
7. The method of claim 4, wherein said substance is an
N-glycosylation pathway inhibitor.
8. The method of claim 7, wherein said inhibitor is a
.beta.1-6-N-acetyl glucosaminyl transferase inhibitor.
9. The method of claim 7, wherein said inhibitor is swainsonin.
10. The method of claim 4, wherein said substance is a galectin-3
ligand.
11. The method of claim 10, wherein said galectin-3 ligand is
lactose.
12. The method of claim 10, wherein said galectin-3 ligand is an
oligosaccharide which comprises an N-acetyl-lactosamine motif.
13. The method of claim 12, wherein said oligosaccharide is
N-acetyl-lactosamine.
14. The method of claim 1, wherein said T lymphocyte is specific
for a complex of an MHC molecule and a cancer associated
peptide.
15. The method of claim 1, wherein said activity is target cell
lysis.
16. A method for determining if a substance modulates activity a T
lymphocyte, comprising determining distance between a TCR molecule
and a CD8 molecule on said T lymphocyte prior to contact with said
substance, followed by determination of said distance after said
contact, a difference there between being indicative of a substance
which modulates activity of said T lymphocyte.
17. The method of claim 16, comprising FRET analysis.
18. Composition useful in modulating T cell production comprising
at least one peptide which binds to an MHC molecule and a substance
which changes distance between a T lymphocyte receptor and
co-receptor to a point sufficient to modulate T lymphocyte
activity.
19. Composition useful in modulating T cell production comprising
at least one vaccine material and a substance which changes
distance between a T lymphocyte receptor and co-receptor to a point
sufficient to modulate T lymphocyte activity.
20. The composition of claim 19, comprising a full length, tumor
rejection antigen precursor protein.
21. The composition of claim 19, comprising a recombinant virus
which expresses all or a part of a tumor rejection antigen
precursor.
22. The composition of claim 19, further comprising an adjuvant.
Description
RELATED APPLICATIONS
[0001] This application claims priority of provisional application
Ser. No. 60/926,265 filed Apr. 26, 2007, and incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods for modulating activity of
T lymphocytes, such as CD8.sup.+ T lymphocytes, or "CD8TLs". More
specifically, it relates to treating T lymphocytes with low or no T
cell activity with one or more compounds which act to bring two
components critical to T lymphocyte activity, the T cell receptor
(TCR), and co-receptor CD8 in proximity to each other sufficient to
increase or restore T lymphocyte activity.
BACKGROUND OF THE INVENTION
[0003] Study of the immune system has shown it to be an extremely
complex one, requiring countless signals in order to function
optimally. See, e.g., Janeway, Quant. Biol., 54:1-4 (1989), Paul,
ed., Fundamental Immunology, (4.sup.th ed., 1998), both of which
are incorporated by reference herein.
[0004] An essential feature of an immune response are the
interactions between T lymphocytes and antigen presenting cells
("APCs"). Many cohesive molecules that are found on T cells and
APCs increase during an immune response. Increased levels of these
molecules are believed to be key to the fact that activated APCs
are more effective at stimulating antigen specific T cell
proliferation, than are resting APCs.
[0005] It is thus not surprising that the T cell immune response is
a complex process, involving cell interactions, including those
between T cells and accessory cells, including APCs, and production
of soluble immune mediators. This T cell response is regulated by
various T-cell surface receptors, including, but not being limited
to, the T-cell receptor complex, as well as other "accessory"
surface molecules. Many of these accessory molecules are naturally
occurring cell surface differentiation (CD) antigens defined by the
reactivity of monoclonal antibodies on the surface of cells.
[0006] It is well established that, on CD8TLs, the TCR and CD8
molecules co-participate in the recognition of a complex formed by
an antigenic peptide, and a class I major histocompatibility
complex, or "MHC" molecule. In humans, these MHC molecules are
known as "human leukocyte antigens" or "HLAs." When "HLA" is used
herein, it is to be understood that it is representative of MHCs
generally.
[0007] Details of the aforementioned interaction are well
established. TCRs are dimers, with ".alpha." and ".beta." chains,
and are sometimes referred to as "TCRabs". These molecules interact
with the peptide, and the surrounding grove of the MHC class I
molecule, with a kD generally higher than 3 mM, in syngeneic
responses. The CD8 molecule, also heterodimeric, and sometimes
referred to as CD8.alpha..beta., contacts the a3 constant region of
the MHC class I molecules, with a kD of approximately 100 mM. See,
Arcaro, et al., J. Exp. Med., 194:1485-1495 (2001); Holler, et al.,
Immunity, 18:225-264 (2003).
[0008] When complexes of TCR and CD8 form, the resulting complexes
have affinities for the MHC-peptide complexes that are about
ten-fold higher than the TCR alone. Holler, supra.
[0009] Structurally, CD8.alpha..beta. molecules are located in
lipid rafts, in association with intracellular tyrosine kinase
p56.sup.lck. Arcaro, et al., supra, Arcaro, et al., J. Immunol.,
165:2068-2076 (2000). TCRs are associated with CD3.gamma., .delta.,
.epsilon. and .zeta. chains, and are not located in rafts, but
TCR-CD3 complexes interact closely with CD8-p56.sup.lck complexes
that are found in rafts, so as to induce signal transduction when
binding to antigen. Doucey, et al., Eur. J. Immunol., 31:1561-1570
(2001); Montixi, et al., EMBO J., 17:5334-5348 (1998).
[0010] After the initial binding of CD8TLs to target cells,
additional TCRs congregate at the contact region, referred to as
the "immunological synapse." Synapses mature to form distinct
patterns with TCR molecules located in the center and surrounded by
LFA-1 adhesion molecules, which are in turn surrounded by CD45
molecules. Huppa, et al., Nat. Rev. Immunol., 3:973-983 (2003); van
der Merwe, et al., Semin. Immunol., 12:5-21 (2000). Further, the
microtubule organizing center, or "MTOC" moves to the synapse,
directing release of lytic granules into target cells. Grakoui, et
al., Science, 285:221-227 (1999); Stinchcombe, et al., Immunity,
15:751-761 (2001). During the process, TCR molecules in the synapse
begin internalizing, resulting in a considerable decrease of TCRs
on the cell surface; however, newly synthesized and recycled TCRs
move to the cell surface, and the initial level of TCRs on cell
surfaces is recovered, usually, within 24 hours of antigen
presentation. Valitutti, et al., Nature, 375:148-151 (1995).
[0011] It is also well known that if CD8TLs are stimulated, e.g.,
weekly, with their target antigen in the presence of growth
factors, they can be kept in culture for several months. It has
been observed, however, that their cytolytic activity can diminish
or be lost completely.
[0012] Demotte, et al., Eur. J. Immunol., 32:1688-1697 (2002), the
disclosure of which is incorporated by reference, observed that
CD8.sup.+ T lymphocytes lose their specific cytolytic activity if,
instead of being stimulated weekly with tumor cells, are stimulated
with Epstein Barr Virus ("EBV") transformed B cells which present
the relevant antigenic peptide. The loss of effector function is
always associated with loss of labeling by HLA-peptide tetramers.
While Demotte et al. were not able to identify the reason for this
loss of activity, they did eliminate loss of TCR or CD8 expression
as reasons.
[0013] Galectin-3 is a well known molecule. An exemplary, but by no
means exhaustive listing of the literature on this molecule
includes Dumic, et al., Biophysica. Acta., 616-635 (2006),
Demetriou, et al., Nature, 409:733-739 (2001), both incorporated by
reference, which provide details on the structure of galectin-3, as
well as a role in forming a lattice, with glycoproteins, leading to
restriction in recruitment of TCRs to antigen presentation
sites.
[0014] Also of interest are U.S. Pat. No. 6,680,306, describing how
surgical procedures can be improved by administering a carbohydrate
which selectively binds to a galectin, including galectin-3; U.S.
Pat. No. 6,890,906, which discusses how the use of complex
carbohydrate molecules which bind to galectins, including
galectin-3, can control angiogenesis, and U.S. Pat. No. 6,770,622
which describes a role for galectin-3 in tumor metastasis, and
describes a truncated galectin-3 molecule useful in treating
metastasis. All of these patents are incorporated by reference
herein.
[0015] Nothing in these references, however, points to or suggests
that galectin-3 has a role in the immunological process known as
anergy.
[0016] It has now been found that the diminishing or loss of T
lymphocyte activity is associated with physical separation of TCR
and CD8 molecules. It has also been found that both the proximity
of TCR and CD8 and the activity of the T lymphocytes can be
restored via treatment of the CD8TLs with appropriate
molecules.
[0017] How this is accomplished will be seen from the disclosure
which follows.
DETAILED DESCRIPTION OF THE INVENTION
Example 1
[0018] CD8TL clone A10 which was isolated from a patient who had
been vaccinated with a recombinant poxvirus, ALVAC, containing the
region coding for peptide EVDPIGHLY (SEQ ID NO: 1), which consists
of amino acids 168-176 of MAGE-3. The peptide is presented by
HLA-A1 molecules. The CD8TLs were isolated by ex vivo tetramer
sorting as described in Godelaine, et al., J. Immunol.,
171:4893-4897 (2003)).
[0019] On day 0, samples of CD8TL clone A10 (a total of
3.times.10.sup.5 cells) were cultured, in 24-well plates, in the
presence of 1.times.10.sup.6 irradiated, allogeneic Epstein-Barr
virus immortalized B cells (EBV-B cells), which served as feeder
cells, and were stimulated with 1.times.10.sup.5 irradiated tumor
cells, which had been incubated, previously for 1 hour at
37.degree. C., with 1 .mu.g/ml of peptide, and washed. The culture
medium was IMDM supplemented with AAG, 10% heat inactivated
autologous human serum, and 50 U/ml of IL-2.
[0020] Fourteen days after the stimulation, the CD8TLs were then
stained with complexes of HLA-A0101 folded with SEQ ID NO: 1,
prepared as described by Coulie, et al., Proc. Natl. Acad. Sci.
USA, 98:10290-10295 (2001), incorporated by reference. The
complexes were biotinylated and then labeled with phycoerythrin
("PE"), using known methods. Staining was accomplished by
incubating CD8TLs, for 10 minutes, at room temperature, with 5 nM
of tetramer, and then washing.
[0021] The cells were found to have strong, homogenous binding to
the tetramer and this set of properties is referred to hereafter as
tetramer.sup.high. At that day, the CD8TLs were again stimulated
under the conditions described supra. By the 4.sup.th day, a
homogenous, approximately 10 fold decrease in tetramer staining was
observed (the tetramer.sup.low phenotype). By 7 days, the CD8TLs
had recovered part of the ability to bind tetramer, and had
recovered it completely by day 14.
Example 2
[0022] To study the decrease in tetramer binding discussed supra,
experiments were carried out to determine if decreased surface
expression of one or both of TCR.alpha..beta. or CD8.alpha..beta.
was involved. To test this, cells were incubated with an
anti-CD3.APC fluorescent antibody to identify T cells, washed, and
then incubated for 15 minutes at 4.degree. C. with 1/30 of
anti-CD8.FITC, after which they were analyzed via FACS. To study
TCR.beta. expression, cells were incubated with an anti-CD3APC
antibody to identify T cells, washed, and then incubated for 15
minutes at 4.degree. C. with TCR.beta..FITC, then washed and fixed
with 1% paraformaldehyde in PBS. The levels of surface expression
of TCR.beta., CD8.alpha., CD8.beta., and TCR.alpha. on days 4 and 7
were the same as those observed on day 0.
Example 3
[0023] In addition to the experiments set forth, supra the effector
capability of the T lymphocytes was tested, at various points
during the period in which they were stimulated. Stimulation
conditions are set forth in Example 1, supra. The effector activity
was determined by contacting samples of 5000 CD8TLs with
2.times.10.sup.5 HLA-A1 EBV-B cells which had been incubated for
one hour with 1 .mu.M of the peptide of SEQ ID NO: 1 at 37.degree.
C., and co-culturing the mixed sample, overnight. The amounts of
IFN-.gamma. and IL-2 resulting from the activation of the T cells
were measured in the supernatants of the co-cultured cells via
ELISAs.
[0024] It was observed that the CD8TLs produced IFN-.gamma. and
IL-2 at day 0, with a significant drop at day 4. The ability to
release IFN-.gamma. was regained by day 14 (IL-2 levels were not
measured).
[0025] In parallel, TCR.beta. intracellular LAT molecules, and CD3
zeta molecules were concentrated on the surface at the contact site
with peptide-pulsed cells at day 0, but the CD8TLs failed to
recruit these molecules at day 4
[0026] This pattern of loss, and then recovery of effector
function, cytokine production and tetramer staining was observed
with fifteen other CD8TL clones. The loss of tetramer binding
followed by recovery was observed with CD8TL clone 5 as shown infra
using tetramer concentrations ranging from 1 to 100 nM.
Example 4
[0027] The experiments described in this example show that the time
course of recovery of high tetramer binding is influenced by the
type of antigenic stimulation the CD8TLs receive.
[0028] CD8TL clone 5 is a second clone which recognizes complexes
of SEQ ID NO: 1 and HLA-A1 and lyses cells presenting these
complexes. It is one of the fifteen cell lines referred to
supra.
[0029] Stimulation of this CD8TL clone was carried out in three
different ways. One way was the stimulation with irradiated tumor
cells, referred to supra for clone A10. A second approach was
stimulation with 0.5 .mu.g/ml of phytohemaglutinnin. A third method
used 3.times.10.sup.5 EBV-B cells that were HLA-A1 positive, and
had been pulsed with peptide, as described, supra.
[0030] The same pattern, i.e., strong initial tetramer binding,
followed by a drop, and recovery within about 2 weeks, was observed
when the stimulus was peptide pulsed, tumor cells; however,
stimulation with the EBV-B cells maintained the cells in a
"tetramer.sup.low" binding phenotype for 2 weeks, with
"tetramer.sup.high" binding only recovered after 4 weeks. The same
results were obtained with PHA stimulation.
[0031] In additional studies, the effect of frequency of
stimulation was tested, by stimulating the clone 5 cells, 4 times,
at 3 day intervals. This maintained the CD8TLs in a
"tetramer.sup.low" phenotype for 2 weeks, with another 3 weeks
being necessary to regain the high staining phenotype.
[0032] These data indicate that the duration of tetramer.sup.low
phenotype is correlated to the strength, and duration, of antigenic
stimulation.
Example 5
[0033] The reduced ability to bind tetramers following stimulation
of long term CD8TLs suggested a further set of experiments, to
determine if this phenomenon could be observed ex vivo, i.e. with
lymphocytes collected from the body, as opposed to long term CD8TL
clones.
[0034] Blood cells were obtained from a donor, and mononuclear
cells were isolated via standard methods, after which they were
cultured for 20 days, in complete IMDM medium with 50 U/ml of IL-2,
and one of Epstein-Barr virus peptide GLCTLVAML (SEQ ID NO: 2), at
5 .mu.M, or PHA (0.5 .mu.g/ml), or an irrelevant peptide. The
mononuclear cells were HLA-A2 positive, and SEQ ID NO: 2 is known
to bind with HLA-A2.
[0035] Following the single stimulation, cells were stained with PE
labeled tetramers, containing SEQ ID NO: 2, as described supra.
[0036] In the results which follow, it will be seen that during the
first week, there was almost no tetramer staining, regardless of
the stimulation; however, review of the results secured 20 days
after stimulation shows that selective proliferation did in fact
occur.
Example 6
[0037] As discussed supra T lymphocytes which express both CD8 and
TCR molecules on their surface in normal amounts fail to engage and
fail to bind HLA-peptide tetramers at one stage of their
stimulation cycle. In view of this, studies were carried out to
determine the distribution of these molecules on cell membranes of
both "tetramer.sup.high" and "tetramer.sup.low" binding
subpopulation of clone A10. To describe these subpopulations
briefly, the "tetramer.sup.high" subpopulation had not been
contacted with antigen for 15 days, while the "tetramer.sup.low"
subpopulation had been contacted with peptide pulsed cells, 4 days
before the study.
[0038] Cells were fixed to cover slips, and then stained with a
combination of an anti-TCR.beta. antibody, and an "Alexa-568"
coupled secondary antibody, which provides a red stain, and a
second combination of an anti-CD8.alpha. antibody and a secondary,
"Alexa488" coupled antibody, which provides a green stain. Standard
assay conditions were used.
[0039] On most cells of the "tetramer.sup.high", population, the
molecules co-localized, appearing as a unique yellow ring. The
"tetramer.sup.low" subpopulation, CD8.alpha. molecules located on
an outer ring on the equator, while TCR.beta. located on an inner
ring, indicating segregation of the molecules.
Example 7
[0040] Given the apparent co-localization of the co-receptors on
tetramer.sup.high cells, it was of interest to determine if they
were close enough to cooperate with respect to tetramer binding. To
determine this, "fluorescence resonance energy transfer" or "FRET"
techniques were used.
[0041] To elaborate, samples of 2.times.10.sup.5 cells were washed
in Hank's medium that had been supplemented with 1% human serum,
and were then resuspended, in 50 .mu.l of medium, which contained
either an anti-TCR.beta.-PE antibody (the "donor" antibody), or an
anti-CD8.alpha.-Alexa647 antibody (the "acceptor" antibody), or
both. The former was diluted at 1/20 and the latter, at 1/300.
[0042] Samples were incubated for 30 minutes at 4.degree. C.,
washed, and fixed with paraformaldehyde (2% w/v in PBS). Analysis
was carried out on a FACS system, and transfer of fluorescence was
calculated as FRET units, in accordance with Doucey, et al., J.
Biol. Chem., 278:3257-3264 (2003), incorporated by reference. A
FRET unit is defined as:
[E3.sub.both-E3.sub.none]-[(E3.sub.A647-E3.sub.none).times.(E2.sub.both/-
E2.sub.A647)]-[(E3.sub.PE-3.sub.none).times.(E1.sub.both/E1.sub.PE)]
[0043] E1 is the fluorescence detected at 580 nm, following
excitation at 488 nm. E2 is fluorescence detected at 670 nm,
following excitation at 630 nm, and E3 is fluorescence detected at
670 nm following excitation at 488 nm.
[0044] After the molecules are excited at the donor level, transfer
of energy from donor to acceptor happens only if the distance
between two molecules is less than 10 nm, with energy transfer
being measured by emission at the acceptor wavelength.
[0045] Substantial transfer of energy was observed only with the
"tetramer.sup.high" population shown that, on this subpopulation of
CD8TLs, the CD8 and TCR molecules are, in fact, close enough to
play their co-receptor role in binding HLA-peptide complexes.
Example 8
[0046] The results obtained in the prior examples suggest that CD8
molecules are segregated from TCR molecules, on the
"tetramer.sup.low" subpopulation of CD8TLs. This implies that some
anchoring activity must cause this.
[0047] Previously, Foti, et al., Proc. Natl. Acad. Sci. USA,
99:2008-2013 (2002), showed that p56.sup.lck anchors CD4 in lipid
rafts, localized on microvilli. The p56.sup.lck molecule has also
been shown to bind CD8.alpha.. Barber, et al., Proc. Natl. Acad.
Sci. USA, 86:3277-3281 (1989). It is also known that Zn is
essential for both binding events. See, Lin, et al., J. Biol.
Chem., 273:32878-32882 (1998). It was thus proposed that if
p56.sup.lck anchors CD8.alpha. at a distance away from TCR in the
"tetramer.sup.low" subpopulation, if CD8.alpha. molecules were
released, a zinc chelator could in fact restore the proximity of
the two relevant molecules and their ability to bind tetramers.
[0048] To test this, cells of CD8TL clone A10 were collected, on
days 0 and 4, after stimulation with HLA-A1.sup.+, MAGE-3.sup.+
tumor cells. Mean fluorescence indices were determined for both the
high and low staining populations, on days 0 and 4. FRET staining
was carried out, as described supra, with cells that were either
incubated with no test substance, or which had been incubated for 2
hours, on day 4, with one of o-phenantholin (30 .mu.M), a known Zn
chelator, lactose, (100 mM), LacNAc (1 mM), or sucrose (100 nM).
The rationale behind the incubation with lactose, LacNAc, and
sucrose is explained in Example 11. A final sample was incubated
with swainsonin (0.5 .mu.M) on day 0, before the cells were
stimulated.
[0049] The cells were then washed, and analyzed for all of tetramer
staining, proximity of the molecules via FRET, and IFN-.gamma.
production following antigenic stimulation.
[0050] It was indeed observed that when the "tetramer.sup.low"
subpopulation was treated with o-phenantholin, it regained FRET
exchanges between the 2 molecules, the ability to bind tetramers,
and the ability to release IFN-.gamma. when stimulated with
antigen.
Example 9
[0051] It has been proposed by, e.g., Demetriou, et al., Nature,
409:733-739 (2001), and Morgan, et al., J. Immunol., 173:7200-7208
(2004), that N-glycosylated, TCR molecules could be part of a
lattice formed by surface glycoproteins, with extracellular
galectin-3. This trapping of the TCR in a lattice could be
responsible for the anchoring of the TCR at a distance of the
CD8.
[0052] Galectin-3 binds with high affinity to N-acetyl lactosamine
("LacNAc") motifs, which are located near the end of branched,
sugar structures, that are bound to asparagine residues. Ahmad, et
al., J. Biol. Chem., 279:10841-10847 (2004) show that galectin-3
favors lattice formation, because it forms homopentamers. The
relevant branched sugar structures are built via a series of
enzymes in an N-glycosylation pathway and
.beta.1-6-N-acetylglucosaminyl transferase V ("MGAT5" hereafter),
modifies branched structures to allow other enzymes to add the
LacNAc motifs.
Example 10
[0053] In experiments which followed up on those described supra,
FRET analysis was carried using one of (i) anti-TCR.beta.-PE or
anti-galectin-3 PE (as donor), and one of (ii) anti-CD8.beta.-Alexa
647, or anti-TCR.beta.-Alexa 647 (as acceptor) and as described
supra. Following staining, cells were fixed and analyzed, via flow
cytometry. Staining was carried out on cells at day 0, and 4 days
after stimulation, and both the "tetramer.sup.high" and
"tetramer.sup.low" subpopulations were tested.
[0054] Co-localization of galectin-3 and TCR were found on the
"tetramer.sup.low" subpopulation of CD8TLs, but not on the
"tetramer.sup.high" subpopulation. No proximity was found between
CD8 and galectin-3 on either population.
Example 11
[0055] In the examples discussed supra, incubation of CD8TLs with
lactose or LacNAc was described. These are both known, galectin-3
ligands, and it was thought that this incubation might serve to
release LacNAc bearing surface proteins, such as TCRs, from the
lattice structure.
[0056] The results indicated that, as compared to untreated cells
of "tetramer.sup.low" phenotype, the cells which had been treated
with either lactose or LacNAc showed higher FRET efficiencies,
which indicated that there was greater TCR-CD8 localization. They
also showed higher degrees of tetramer staining, and a higher
ability to release IFN-.gamma. when subjected to antigenic
stimulation.
[0057] Sucrose is not a galectin-3 binder, and treatment with it
had no effect. Swainsonin is a known inhibitor of
.alpha.-mannosidase II, which is also involved in the
N-glycosylation pathway referred to supra. When it was added to
CD8TL medium prior to restimulation, cells showed higher tetramer
staining (at day 4), as well as higher FRET efficiency and a higher
ability to release IFN-.gamma. when restimulated.
Example 12
[0058] These experiments were designed to determine if TCRs and CD8
molecules colocalize on TILs. It has been reported that solid
tumors in humans, as well as tumor ascites, are infiltrated by T
cells. See, e.g., Ionnides, et al., J. Immunol., 146:1700-1707
(1991); Yannelli, et al., Int. J. Cancer, 65:413-421 91996); Zorn,
et al., Eur. J. Immunol., 29:602-607 (1999). In the case of
melanoma, detail studies revealed that most of these TILs are
ineffective in situ, i.e., tumors clearly progress notwithstanding
the presence of the TILs. See, Germeau, et al., J. Exp. Med.,
201:241-248 (2005); Lurquin, et al., J. Exp. Med., 201:249-257
(2005). In studying TILs from solid tumors, since the nature of the
HLA-peptide complexes was not known, tetramer staining was not a
viable option; however, the FRET analysis described supra does
provide a viable methodology for these experiments.
[0059] A standard depletion strategy was used to isolate CD8.sup.+
T cells from a breast carcinoma metastasis, ovarian carcinoma
ascites, blood, and a kidney allograft that had been resected
during a chronic rejection process.
[0060] The breast and ovarian carcinoma ascites provided enough
CD8.sup.+ TILs to carry out the assay, and the FRET analysis of the
TCR and CD8 proximity showed no significant transfer of energy
between TCRs and CD8, showing a lack of colocalization. This was in
strong contrast to CD8.sup.+ T cells from blood, which showed a
clear energy transfer. Energy transfer was also apparent with
CD8.sup.+ T cells isolated from the kidney allograft resection. The
transfer was equivalent to that on control tetramer.sup.high CD8TL
clones. The conclusion from this is that TIL anergy is associated
with a lack of colocalization of TCR and CD8.
Example 13
[0061] The question that was then posed was whether the functions
of CD8TLs could be restored by appropriate treatment.
[0062] These experiments tested the impact of N-acetyllactosamine
(LacNAc) on colocalization. Gastric carcinoma ascites were screened
for CD8.sup.+ TILs, after they had been incubated for various time
periods at 37.degree. C., with 1 mM of LacNAc, and washed. As a
control, the line CD8TL A10 was used, two weeks after its last
antigenic stimulation. It was found that, after 5 hours of
treatment with LacNAc, the TILs had recovered the same level of TCR
CD8 FRET as had been observed on tetramer.sup.high CD8TLs.
[0063] Not surprisingly, when assays for IFN-.gamma. were carried
out with TILs from ovarian cancer ascites, the TILs did not produce
IFN-.gamma. even after overnight stimulation with a mixed
population of tumor cells, B cells and macrophages (from the same
ascites), or with allogenic, EBV-B cells. The TILs showed low
responsiveness to CD3/CD28 stimulation; however, an overnight
incubation with 1 mM LacNAc boosted IFN-.gamma. response to antigen
stimulation by more than 10 fold. This was also the case for
release of TNF-.alpha., IL-4, IL-5, and IL-10. Similar results were
secured with ascites for endometrium carcinoma and ovarian
carcinoma.
[0064] As was noted supra, there was clear TCR CD8 FRET on kidney
infiltrating T cells isolated from the kidney allograft. These T
cells released IFN-.gamma. upon stimulation with allogeneic EBV-B
cells, or beads coated with anti-CD3/CD28 antibodies. The LacNAc
treatment did not result in increased IFN-.gamma. production.
Example 14
[0065] The preceding experiments established that TCR and CD8
co-receptors are separated on tetramer.sup.low cells, and on TILs.
This opened the possibility that they are anchored, at different
locations on the cell membrane, by different molecules.
[0066] Demetriou, et al., Nature, 409:733-739 (2001), and Morgan,
et al., J. Immunol., 173:7200-7208 (2004), have suggested that
N-glycosylated TCR molecules belong to a surface lattice of
glycoproteins, clustered by extracellular galectin-3. It is known
that galectin-3 binds, with high affinity, to LacNAc motifs,
positioned near the end of N-linked branched sugar structures which
are bound to asparagine residues of surface proteins. Lattice
formation occurs as a result of homopentamers formed by galectin-3.
(Ahmad, et al., J. Biol. Chem., 279:10841-10847 (2004).
[0067] If one assumes a TCR molecule is anchored, in a
glycoprotein-galectin-3 lattice, at a distance from a CD8 molecule,
it might be the case that a galectin-3 ligand would release the TCR
upon binding to galectin-3, thus restoring proximity of TCR and
CD8, and the function of a T cell.
[0068] To test this, tetramer.sup.low CD8TLs, collected 3-4 days
after stimulation, as described supra, were treated with LacNAc, a
known competitive binder of galectin-3. Release of IFN-.gamma. was
restored.
[0069] These experiments were repeated testing two other galectin-3
binders, "tri-LacNAc," and an antibody against the N-terminus of
galectin-3. Both of these substances exerted similar effects on the
target CD8TLs.
[0070] Similarly, when cells were treated with neuraminidase, which
is known to cleave sialic acid from many glycans, the effect was
similar to that of LacNAc.
[0071] The experiments described herein, using neuraminidase, were
carried out on human CD8TLs, and the results parallel those
observed by Kao, et al., Immunol., 17:1607-1617 (2005), on murine
cells. It is hypothesized that a sialic acid residue may be
present, near the LacNAc motif of galectin-binding proteins, and
act to increase galectin-3 affinity.
Example 15
[0072] The experiments described supra, using FRET analysis, showed
that there was a lack of colocalization in anergic CD8.sup.+ TILs
isolated from tumors, while the clear energy transfer on CD8.sup.+
blood T cells indicated colocalization had occurred. These results
lead to the conclusion that TIL anergy is in fact associated with a
lack of receptor colocolization. It was confirmed by the fact that,
notwithstanding overnight stimulation with a mixed population of
tumor cells, B cells, and macrophages collected from the same
ascites, or with allogenic EBV-.beta. cells. IFN-.gamma. was not
produced. They also exhibited a very low response to anti-CD3/CD28
stimulation.
[0073] When these anergic TILs were incubated overnight with 1 mM
LacNAc, their IFN-.gamma. response increased more than 10 fold.
Similar results were obtained with TILs treated with TriLacNAc, and
neuraminidase.
[0074] Different treatments were not, however, unspecific.
Functional kidney infiltrating lymphocytes, and functional CD8TL
3.2, collected fifteen days post stimulation, did not show an
increased ability to release IFN-.gamma., notwithstanding the
treatment.
[0075] The foregoing disclosure sets forth various features of the
invention, which relates to methods for modulating activity of a
CD8 T lymphocyte ("CD8TL" hereafter, or "CD8TLs" when the plural is
used), by increasing or decreasing it. "Increasing activity" as
used herein, refers both to augmenting pre-existing activity of a
CD8TL, even if that activity is at a low level, and to activating a
CD8TL which is showing no activity. This is accomplished by
contacting the CD8TL with a substance that enables two components
necessary for T lymphocyte activity, i.e., the CD8TL receptor and
co-receptor, to change the distance between each other. If the
modulator that is used is an activating agent, it reduces the
distance between the receptor and the co-receptor such that they
interact in a fashion characteristic of active and functional T
lymphocytes, and vice versa for an inhibiting agent. Conversely,
"decreasing activity" refers to lowering pre-existing activity of a
T lymphocyte, or even eliminating it. Activity is also the
production of cytokines such as IFN-gamma.
[0076] The co-receptors are CD8 molecules and the receptors are T
cell receptor molecules. The method involves contact with one or
more substances which release one or both molecules from lattices
to which they are bound or strengthen that interaction.
[0077] The nature of these "modulators" will vary, as the nature of
lattice binding or anchoring to the cell membrane differs for each
receptor and co-receptor, and explained supra. For example, a zinc
chelator, such as o-phenantholin can be used to release a CD8
molecule from the cell membrane which it is anchored.
N-glycosylation pathway inhibitors, such as
.beta.1-6-N-acetylglucosaminyl transferase inhibitors, e.g.,
swainsonin, may be used to impede the formation of the TCR-galectin
lattice. When release of the T cell receptor from the lattice is
desired, a galectin ligand, such as a galectin-3 ligand, e.g.,
lactose, or an oligosaccharide which comprises an N-acetyl
lactosamine motif, such as N-acetyl lactosamine molecule per se can
be used.
[0078] It should be noted that the materials discussed supra are
exemplary, but are not the only examples of substances which will
accomplish the stated purpose of the invention, which is the
modulation of activity of a CD8TL. Determining such modulation is
easily determinable by one of ordinary skill in the art.
[0079] Exemplary of galectin-3 modulating molecules include
GCS-100, a modified form of citrus pectin, described, e.g., by
Chaunan, et al., Canc. Res., 65(18):8350-8358 (2005), incorporated
by reference. Published U.S. Patent application 2004/0223971, also
incorporated by reference, describes modified pectins and
carbohydrate containing polymers as inhibitors, as well as
antibodies against galectin-3. U.S. Pat. No. 7,230,096, also
incorporated by reference, also discusses carbohydrate derivatives
as galectin-3 inhibitors. U.S. Pat. No. 6,770,622, cited supra and
incorporated by reference, describes truncated forms of galectin-3,
showing that proteins and polypeptides can serve as inhibitors of
galectin-3 as well. Additional galectin-3 inhibitors embraced by
the invention are described in U.S. Pat. No. 6,680,306,
incorporated by reference, which teaches polymers with side chains
that terminate with galectose and/or arabinose units.
[0080] All of these materials, as well as those described in the
Examples, and substances which function in accordance with the
definition of modulator set forth supra constitute a part of the
claimed invention.
[0081] When increasing the activity of a T lymphocyte, the CD8TL is
preferably one which is specific for a complex of an MHC molecule
and a peptide associated with cancer, however, as CD8TLs target
various peptide/MHC complexes, any CD8TL can be used, regardless of
its specificity. While exemplification is provided for CD8 T
lymphocytes, it is envisioned that the methodology is applicable to
CD4 lymphocytes as well.
[0082] As noted supra, the disclosure provides various ways to
determine if a compound or substance increases the activity of a
CD8TL, and this screening assay feature is also a part of the
invention. FRET analysis, as described supra, is especially
preferred for this aspect of the invention.
[0083] Also a feature of the invention are what will be referred to
as "combination therapies" and compositions that are useful in
these. "Combination therapy" as used herein, refers to the use of a
galectin-3 modulator in combination with one or more peptides which
are presented by MHC molecules and provoke an immune response,
and/or a substance or combination of substances which undergoes
processing to such a peptide. These peptides can be, e.g., tumor
rejection antigen or T lymphocyte peptides, such as, but not being
limited to, MAGE, BAGE, GAGE, NY-ESO-1, PRAME, FRAME, Melan-A, or
other tumor rejection precursor based antigens, as well as other
peptides known to be presented by MHC molecules on the abnormal
cells of the patient being treated. It is well within the skill of
the artisan to determine MHC type of a patient and to choose
appropriate peptide. While so-called "Class I" binding peptides are
preferred, Class II binding peptides may also be used, as well as
combinations. While one or more peptides may be combined with the
modulator, the modulator may also be combined with full length
proteins, such as a so called "tumor rejection antigen precursor,"
as described supra, or some other material which functions as a
vaccine. By vaccine is meant, e.g., a substance or combination of
substance with the immune stimulating effect described supra. Non
exhaustive exemplification of such vaccine materials include full
length TRAPS, as described above, as well as fragments thereof,
which are larger than the TRAs, and smaller than full length TRAPS,
which are processed to one or more TRAs, isolated cells which
present the TRA molecules on their surfaces, mutated viruses which
express the protein in question, but have been modified to be
harmless to the host, including etiolated forms of viruses,
transfected bacteria which have been treated and rendered
non-proliferative, and complexes of TRAs and an MHC or HLA
molecule. These vaccine materials may be combined with an
appropriate adjuvant. These are well known to the skilled artisan,
and need not be repeated here.
[0084] In use, the therapeutic method may involve simultaneous use
of the modulator and at least one peptide or other vaccine material
as well as methods where the modulator precedes, or follows the at
least one peptide or other vaccine material.
[0085] Compositions which are useful in these therapies are also a
part of the invention. In combination therapy, the composition may
include both the galectin-3 modulator and at least one peptide
and/or vaccine material, together with a pharmaceutically
acceptable carrier, which may be, e.g., an adjuvant. The
composition may also include optional ingredients, such as a
molecule which facilitates delivery of the inhibitor to the
cells.
[0086] These compositions may also take the form of multi-component
"kits," where separate portions of the modulator and the at least
one peptide or other vaccine material are provided, in the form of
pharmaceutically acceptable compositions, which are in turn
presented in a kit type container, so that the treating physician,
e.g., can inoculate, or otherwise administer one, then the other
agent.
[0087] Any form of administration of the compositions is a part of
the invention including, but not being limited to, intravenous,
subcutaneous, controlled delivery, oral, intramuscular, sub venous,
and other forms of therapy known to one of skill in the art.
[0088] Other features of the invention will be clear to the skilled
artisan, and need not be set forth here.
[0089] The terms and expression which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expression of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
Sequence CWU 1
1
219PRThomo sapiens 1Glu Val Asp Pro Ile Gly His Leu Tyr1 529PRTHomo
sapiens 2Gly Leu Cys Thr Leu Val Ala Met Leu1 5
* * * * *