~93/14189 ~ PCT/US93/00083
~T~OD FOR D~TERMINING CYT~LYTIC T CELL PRECUR80R8
FIELD OF THE INVENTION
This invention relates to methods for determining precursors
of cytolytic T cells specific for antigens characteristic of
or specific to tumors. More particularly, it relates to a
non-obvious application and modification of the "limiting
dilution" assay to determine the precursors discussed above.
The applications include the ability to monitor therapeutic
regimes and subject response, to particular treatments,
including immune responses.
BAC~GROUND AND ~RIO~ ART
The ability to measure and assay events that occur in an
immunological response is of great interest and importance in
studying the course of a pathological condition or disease.
Where a T-cell response is involved, of course, measurement
and study of T cells is of concern.
T cells constitute a mixture of various types of
lymphocytes, including cytolytic T cells, or "CTLs" as these
will be referred to hereater. CTLs interact with a molecule
presented by their target cell via the ~ cell receptor.
Following the interaction, the CTL causes the target cell to
lyse.
The actual target recognized by the CTL is fre~uently
referred to as an antigen, and this term will be used
hereafter.
Prior work has established that various tumors can and do
present antigens which can be the subject of a T-cell mediated
response, leading to and rejection. Early work by Prehn et
al., J~ Nat. Canc. Inst. 18: 769-778 (1957); Klein et al.,
Canc. Res. 20: 1561-1572 (1960); Old et al., Ann. N.Y. ~cad.
Sci. 101:
80-106 (1962); Kripke et al., J. Nat. Canc. Inst.l 53: 1333-
1336 (1974~, and Van Pel et al., J. Exp. Med. 157: 1992-2001
(1983) have established that the antigens expressed by these
cells, now collectively referred to as "tumor rejection
antigens", exist on murine tumors induced by viruses,
WO93/14189 ~ PCT/~!S93/0~
chemicals, and ultra-violet ,adiation, as well as on
spontaneous tumors. It ~as also been shown that, in some
tumor systems, these tumor rejection antigens induce cytolytic
T cell responses and highly specific CTLs, directed against
murine tumor rejection antigens have been isolated. See
Brunner et ~l., J. Immunol. 124: 1627-1634 (1980). The target
antigen of these CTLs is relevant for recognition by a
syngeneic host, since tumor cells which escape partial immune
rejection in vivo have been found to have masked or to have
lost the antigen CTLs recognize. See ~yttenhove et al., J.
Exp. Med. 157: 1040-1052 (1983). Adoptive transfer with
cloned CTLs can eradicate tumor cells in animals bearing large
tumors. See Kast et al., Cell 59: 603-614 (1989). The murine
tumor P815 encodes a tumor rejection antigen, the gene for
which has been identified and isolated, as per Van æe Eynde et
al., J. Exp. Med. 173: 1373-1384 (1991). It was found that
this gene is identical to one that can be expressed by normal
mouse cells, but has little or no expression in normal adult
mouse tissues.
Studies extended to humans have found that mixed lymphocyte
tumor cell cultures ("MLTCs") frequently generate responder
lymphocytes which lyse autologous tumor cells without lysing
natural killer ("NK") cells, autologous EBV transformed B
cells or autologous fibroblasts. See Anichini et al., Int. J.
Cancer 35: 683-689 (1985). The response has been studied for
melanomas in MLTC, using peripheral blood monocyte cells
~PBMCs) or tumor infiltrating lymphocytes (TILs) as reported
by Mukherji et al., J. Exp. Med. 158: 240-245 (1983); Knuth et
al., Proc. Natl. Acad. Sci. 86: 2804-2808 (1989~; Herin et
al., Int. J. Canc. 39: 390~396 (1987~; Topolian et al., J.
Clin. Oncol. 6: 839-853 (198~). MTLCs derived from PBMC
generally contain responder cells which exert lysis on both
tumor cells and on NK targets, when analyzed after two weeks
of culture. After an additional two or three weeks, lytic
activity on tumor cells increases, and that on NK targets
disappears. See Herin et al., supra. It has been possible to
derive CTLs from MLTC responders which seem to be completely
~93/14189 2 1 2 3 `1 2 ~1 PC~rtUSQ3tO0083
specific for tumors. The antigens recognized on tumor cells
by these CTLs do not appear to be cultural artifacts, as they
have been found to be present on fresh metastatic tumor tissue
cells as well. See Mukherii, supra. Herin, supra; Knuth,
su~ra. Panels of autologous CTLs permitted identification of
four different stable antigens on human melanoma. See Van den
Eynde et al., Int. J. Cancer 44: 634-640 (1989). Analysis of
70 additional CTLs from the patient on which the antigens were
identified shows that all major stable antigens recognized by
CTLs were in this set of four.
Assessment of the role of CTLs specific to tumor cells
requires an ability to assay the fre~uency of their precursors
in patients. This would permit the evaluation of, e.g.,
therapy protocols such as immune therapy approaches, and how
these are affecting the CTL precursors (CTL-Ps), if at all.
The classic way to determine components of T cells is via
a limit.ing dilution assay. This well known technique is
described, e.g., by Sharrock et al., Immunol. Today 11(8):
281-285 (1990~, the disclosure of which is incorporated by
reference. These assays are designed to determine the
frequency of a particular type of cell in a mixed population.
A particular response is studied, in a number of different
samples, and negative responses are used for further
calculations. Negative responses are used because it is not
immediately possible to know if a positive response can be
associated with a particular cell. Essentially, a group of
test cells, the "responder cells" are mixed with a second
group, the "stimulator cells", and any reaction is then
studied. The responder cells are used in a known or defined
number, which can vary determining on the particular protocol
being used. These assays have been carried out for CTLs, as
reported by Sharrock, who discuss the importance of choice of
an appropriate target cell, generally blast cells stimulated
by concanvilin A or phytohemagglutinin. Epstein Barr virus
transformants can also be used, if mixed with other targets.
The technique, while useful, is described as being in its
infancy by Sharrock et al. Thisreference does teach the use
WO93/14189 ~;l;'S t~/1 PCT/US93/00~
of PBNCs as responder cells, but shows a limited number of
choices as stimulators.
It has now been found that one can in fact assay for CTL-
P cells which are specific to tumor antigens. This type of
assay is not ~hown by the art, and is not suggested by the
references which constitute the prior art. Thus, a method for
a~saying for CTL-Ps using a limiting dilution assay is the
subject of the invèntion. The applications of this include
the monitoring of subject response to therapeutic treatments,
as explained infra.
BRIFF DE8CRI~TION OF T~E FIGURE~
Figure l is a depiction of lytic patterns and criteria for
their classification.
Figure 2 presents levels of tumor cell lysis via limiting
dilution microcultures and Poisson analysis of data.
Figure 3 shows tumor cell lysis via limiting dilution, when
competing K562 is added.
Figure 4 shows results obtained for frequencies in anti-tumor
lytic effectors, in presence and absence of K562.
Figure 5 presents frequencies of lytic effeGtors among PBMCs,
CD4+ and CD8+ cells.
DETAILED DE8CRIPTION OF PREFERRED EMBODIMENTS
The examples that follow use different reagents and cells
in a variety of contexts. To simplify the presentation,
certain explanations are provided here.
When "medium" is used, unless otherwise noted, it is
Iscove's medium, supplemented with 0.55 mM L-arginine, 0.~4 mM
L-asparagine, 1.5 mM L-glutamine and 5xl0 5 2-mercaptoethanol.
Human serum, abbreviated hereafter as "HS", refers to pooled
types A, B and O serum obtained from healthy donors. The HS
had been decomplemented by treatment at 56C for 30 minutes,
partially delipified via centrifugation (45 minutes, 17,000g)
filtration and sterilization.
Fetal bovine serum is abbreviated as "FBS".
Interleukin-2 and Interleukin-4 are abbreviated as IL-2 and
- 93/14189 ;'. 1 ,? ~ .i PCT/US93/~X~3
IL-4, respectively. In the following experiments, the
recombinant, human form of the interleukins was used, although
other forms would be expected to work as well. The
concentrations used were 30 U/ml (IL-2), and 5 U/ml (IL-4) in
all experiments. One unit/ml of
IL-2 is defined as the concentration supporting half of
maximal proliferation of CTLL-2 cell line. One unit/ml of IL-
4 is that concentration which yields 50% maximal proliferation
of human T cells previously treated with phytohemagglutinin-
A ("PHA").
The cytokine "gamma interferon" is abbreviated as "IFN-~".
`~AMPLE ~
Peripheral blood mononuclear cells (PBMCs) or purified
subsets were obtained from patients. The patient samples were
subjected to density gradient centrifugation, and were
cryopreserved prior to use in the limited dilution assays
described infra. To separate CD4+ and CD8+ T cells from these
samples, flow cytometric sorting was used, employing labeled
anti-Leu 3 antibody (fluorescein), and anti-Leu 2 antibody
(phycoerythrin). The particulars of the sorting are not of
special relevance to the invention, and purifi~ation of PBMCs,
CD4+ and CD8~ can easily be carried out via standard methods~
Sorted subpopulations were always more than 97.5% pure.
.
EXAMPLE 2
Limiting dilution assays were set up using various
quantities of either-PBNCs or the subsets described supra.
The numbers ranged from about 200 to 10,000.
The particular amount, i.e., the defined number of
PBMCstsubset chosen were seeded in 96 V~bottom microwells,
together with 10~ irradiated autologous tumor cells (i.e.,
tumor cells taken from the same patient as the PBMC source)~
The tumor cells had previously been used to establish cell
lines, and the irradiated cells were taken therefrom.
Irradiation was carried out using a Cs source (10000 rads).
` Irradiated tumor cells served as stimulator cells. The
medium, as defined supra used to culture the mixture was
WO93/14189 ~ PCT/US93/0~ ~
supplemented with l0~ HS and IL-4 as described su~ra. The
wells were centrifuged for three minutes at l00g and incubated
at 37OC in 8% Co2. On the third day of incubation, IL-2 as
described su~ra was added.
On the seventh day of culture, fresh medium (l00 ul) was
added, together with IL-2, IL-4, and 104 additional irradiated
cells.
On the fourteenth day of culture, l00 ul of medium was
discarded, the cells were transferred into flat bottom
microwells, and IL-2, IL-4, and irradiated cells as above were
added.
Over the course of culturing, the rate of proliferation of
the CTL precursors in the sample were measured using
radiolabeled thymidine and conventional methods. On those
days, four aliquots of 40 ul of the cells were transferred
into microwells and lytic activity was assayed. At the same
time, the remaining samples were restimulated by adding 160 ul
of ~edium, again containing the described IL-2, IL-4, and
irradiated cells. At least l00 cultures were carried out for
each number PBMC.
Stimulation of the type described suPra led to proliferation
of sufficient cells to allow assay of lytic activity against
tumor cell.
~X*MP~E 3
To determine the lytic activity of the proliferated cells,
these were tested with autologous tumor cells in the manner
described below.
The autologous tumor cells to be used were preincubated for
48 hours in medium containing 50 U/ml IFN-~. This enhances
expression of histocompatibility molecules and adhesion
molecules. When non-active n~tural killer cells ("K562",
discussed infra) were added, these were not so treated~ IFN-
~ does not modify susceptibility of these cells to lysis via
NK-like effectors.
The tumor cells were labeled via suspending them at 107
cells/ml in medium supplemented with 10% HS, and i~cubated at
37C for 60 minutes, using 200m 5lCr Ci/ml. The labeled cells
f~ 1 2 ~'J 1 ;'~, '',~
~93/14189 PCT/US93/~083
were washed three times with medium and 2% HS, and were then
suspended in medium at 104 cells/ml. Following this, 60 ul of
medium augmented with 2% HS or with an additional 5xl0~ K562
cells/well were added to wells containing the previously
stimulated cells. After one hour of this incubation (37C),
l000 tumor cells were added per well in l00 ul of medium. The
mixtures were centrifuged (4 minutes, 200g) and then incubated
for 4-5 hours at 37C in 8% CO2 atmosphere. Aliquots of
supernatant (l00 ul) were collected and 5lCr specific release
calculated. The following formula was used:
%RELEASE - (ER-SR)xl00(MR-SR)
Where: ER is experimental 5lCr release, SR - spontaneous
release (i.e., release by cells incubated in medium alone),
and MR the "maximal release", i.e., that obtained by
incubating the tumor cells in 0.15% TRITON X-l00. SR never
exceeded 15% of MR.
Figure 2 shows results secured using samples taken from two
patients (LB-33 & LB-30), using varied numbers of PBMCs and
the percentage lysis of melanoma cells taken from the patient.
High degrees of lysis were observed in many microcultures. In
control cultures, i.e., those where no tumor ~iells, and hence
no antigen was presented, there was no lysis.
As the number of PBMCs decreased, the fraction of posi~ive
microcultures also decreased, but the level of lysis did not
decrease more than expected.
Analysis of the lysis pattern showed that a clear bimodal
distribution allowing easy discrimination between positives
and negatives did not exist. This is consistent with an
earlier observation that different effector cells display
different rates of proliferation, thus affecting the degree of
lysis.
When the percentage of microcultures devoid of lytic
activity is plotted as a function of PBMC number and using
zero order terms of Poisson distribution, the logarithm of the
fraction of negative wells decreased linearly when the
quantity of PBMC increased. Figure 2 shows that this is true,
regardless of whether 5% or l0~ lysis was chosen as threshold
WO93/14t89 PCT/US93/~3
~l~Si~"l 8
for positivity.
~AMP~E 4
In the past, stable lytic clones derived from responder
cells have been reported to include anti-tumor CTLs, which
show little or no lysis of the cell line K562, which is a
natural killer (NK) cell target, as well as other clones which
do lyse K562. (K562 is a human chronic myelogenous leukemia
cell line, freely available from the American Type Culture
Collection, e.g., under Accession Number ATCC CCL 243). ~his
cell line has been described, e.g., at J. Nat. Canc. Inst. S9:
77-83 (1977), as a highly sensitive in vitro target in NK
assays. The K562 specific lytic clones are referred to as "NK
like", and it was important to determine if these were
present.
To do this, the limited dilution materials described suDra
were assayed for affect on K562, the NK target. Assays were
run, in both the absence and presence of a 50 fold excess of
non-viable K562 relative to the number of tumor cells in the
sample. The assays used the chromium release methodology
discussed suPra. The result showed that stimulated PBMC
samples fall into one of four patterns:
l. Some microcultures lyse tumor cells, but do not lyse
K562. In such cultures, competition by unlabeled K562 never
significantly reduced the tumor cell lysis.
2. Both tumor cells and K562 were lysed. In competition
with "cold" K562, the lysis of labeled` K562 was nearly
abolished, but tumor cell lysis was not. Such cultures show
a mixture of tumor specific, and NK like clones.
3. Both tumor and K562 cells lysed, where competition with
cold K562 abolishes all lysis. Su~h cultures appear to
contain at least one NK like clone.
4. Lysis of only K562. Such cultures clearly contain NK
like clones, but not tumor specific cells.
The sum of "l", "2" and "3" corresponds to the total amount
of anti-tumor lytic clones as described su~ra. Patterns "l"
and "2" indicate anti-tumor CTLs, and can be used to determine
~3/14189 ~ 1?,~,?~ 2 ~ PCT~US93/~
the frequency of CTL precursors. Table 1 summarizes the
classification into one of the four types listed su~ra~ The
abbreviation "LDA" used therein stands for "limiting dilution
assay".
In the presence of competing K562, a decrease in PBMC number
did not reduce tumor cell lysis more tban what would be
expected from loss of cultures containing multiple CTL clones.
These results are shown in Figure 3.
<IMG>
093/14189 2 ~ 2 ~ 1 PCT/US93/~83
'11
~XAMPL~ 5
The clones considered to be anti-tumor CTLs were examined
for specificity. This was done by testing lytic cultures on
various targets. Table 2, which follows, presents one set of
these data, where PBMCs of a patient (LB-33) were tested
against autologous tumor cells, K562, autologous PHA blasts,
and tumor cells from patients LB-30 and LB-34. These cells
were taken from cell line cultures as discussed su~ra. The
lysis percentage was determined 28 days after setting up a
lytic assay as described su~ra, using 1333 PBMCs per well. In
the case of PHA-blasts, lysis was checked using ~butologous
PBMCs activated for lO days with ~000 U/ml of
IL-2.
WO93/14189 ~ ~ v '1 2 ~ 12 PCI`/US93too(l~
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~93/14189 ~1~3 12~ PCT/~593/00083
EXAMPLE 6
Frequency of anti-tumor CTL precursor cells in various
patients was evaluated. Limiting dilution cultures were set
up using PBMCs, as described supra, using all of the indicated
materials. Lytic activity was measured on the day indicated
in the panels of Figure 4 ("d20", e.g.), and frequency of
lytic effectors calculated. Anti-tumor and total of all lytic
effectors are presented.
The relationship between number of PBMCs added and logarithm
of fraction of microcultures without anti-tumor CTLs appear
linear. This supports the point raised suPra, that tumor
specific lysis appears to result from activity of single T
lymphocyte clones. Large variations were observed in
different patients, both in frequencies of all tumor cell
lysing cells, and in precursors of anti-tumor specific CTLs.
The ratios between these two frequently showed large
differences as well.
EXAMPL~ 7
Analysis was carried out to determine frequency of NX-like
effector clones, as detected around day 20.of the limiting
dilution assays, as well as anti-tumor CTL precursor
frequencies. These are shown in following Table 3.
WO93/14189 2 12 S i 2 ~1 14 PCI`/US~3/00' ~~
5T~ble 3. ~t~ti-~umoral CTL-P frcquencics of diffcrcn~ ~cl~om~ p~icnls.
~:rcqucncics of
Pa~ient E~cp. Day spc~ efleclor ce~ls
.
LB-33 11 20 I~229(1 1/860
2 18 l/lIB0 1/770
22 ~ 90 112560
29 1/2310 118350
3 2û ~L~;2Q 11780
4 ~8 1/910 1/102~
S 20 1/1440 1/610
28 112100 ~J5û90
LB-34 21 116310 I/glO
L8-2~ 18 117120 . 1/250
MZ-2 1~ 26 1~1~3400 1/980
2 22 1/9640 1/2~60
LB-30 1~ 2~ 1/108û0 1/420
2 27 ~QQ 1/20S~
L~ 17 31 1/17900 1/830
LG-2 20 ~ 1/400
~93/14189 2 1;?.(~Q)!t~ PCT/US93~00083
This table shows activity against autologous tumor cells, both
with and without non-viable K562. The microcultures of
pattern "l" or "2" were used to estimate CTL precursor (CTL-
P) frequencies, and patterns "2", "3" or "4" used to estimate
NK-like effector cell frequencies.
It is noted that constant CTL-P frequencies were observed
even though there was some decrease with time in the number of
microcultures with activity. The reason for this is that
under the conditions used, most CTL clones do not proliferate
indefinitely, most probably because of the absence of feeder
cells.
Cultures with NX-like activity decreased more severely
between days 14 and 30, which is consistent with prior
observations by Hérin et al., Int. J. Cancer 39: 390-396
(~987).
E8AMPLE 8
Prior experiments leading to isolation of anti-tumor CTL
clones by limited dilution around day lS showed both CD4+ and
CD8+ cells, but the vast majority of anti-tumor CTLs were
CD8~. Studies were carried out to determine if this was
because culture conditions favor production- of CD8+ cells~
Using the sorting meth~dologies described su~ra, it was found
that most CTL-Ps are CD8~ '
Several features of the examples presented supra merit
comment. Firstt the microcultures did not use feeder cells in
the cultures~ For many patients, it is difficult to obtain
the autologous PBMCs that are desirable feeder cells in
sufficient a~ounts. It was also desired to present a
generally applicable limiting dilution assay. In experiments
performed by the inventors and not reported herein, when
autologous, irradiated PBMCs were used as feeder cells, an
increase in anti-tumor CTL frequency was observed, but t~is
was coupled to higher NK-like frequency.
The experiments using the NK inhibiting material, i.e., non-
viable X562 cells permits distinction between anti-tumor CTLs
and NK like effectors which also lyse tumor cells. The NK
like effectors are inhibited by binding to the K562 cells,
WO93/14189 PCT/US93/000
~ i 2~ 16
which, since they are non viable, do not show 5lCr release.
Other methods to eliminate the effect of NK and/or NK like
cells in the mixture, including other NK target ceils, can
alio be used.
The frequencies of CTL-Ps specific for antigens
characteristic of tumor cells ranged from l/33000 to l/900
PBMCs. The latter number, obtained with patient LB-33, may
result from an autoimmune response against a tumor. The
numbers observed should be compared to those secured by
Borysiewicz et al., Eur. J. Immunol. l8: 269-275 (1988) of
l/5000-20,000 for CMV, by Schmid et al., J. Immunol. 140:
3610-3616 (1988) of l/4000-8000 for HSV, or by Hickling et
al., J. Virol. 61: 3463-3469 (1987), for varicella zoster of
l/1600-90000. Sharrock, Immunol Today ll: 281-286 (l990)
discusses alloreactive frequencies of l/500-500000.
The invention thus teaches a method by which cytotoxic T
cell precursors (CTL-Ps) specific for antigens characteristic
of tumor cells can be determined via a limiting dilution
assay. In the methodology, a defined number of peripheral
blood mononuclear cells, which contain the CTL-PS, are
contacted to an antigen which is characteristic of, charge
for, i.e., is particularly associated with, a tumor type or
types. This results in a mixture of materials which is then
cultured. The stimulation from the tumor antigen causes the
CTL-Ps to develop into the actual cytolytic T cells. This
development is monitored via monitoring the lysis of tumor
cells either in or added to the mixture.
The number of PBMCs used may vary, but preferably somewhere
between about 200 and about l0000 are used per assay. The
PBMCs may be added in a mixed sample~ or as a pure culture of
PBMCs.
The antigen characteristic of the tumor is ideally added in
the form of a tumor cell, generally non-viable. The tumor
cell can be rendered non-viable via, e.g., irradiation. The
tumor cells still present the antigen on their cell surfaces,
an~ a proliferation linked reaction with CTL-Ps follows. The
antigen can also be added, e.g., in pure form, but this is not
~93/14189 2 ~ 2 8 ~ PCT/US93/00083
17
the preferred mode.
It is especially preferred to administer an autologous
sample of tumor cells - i.e., tumor cells taken from the same
subject as the PBMCs. Ideally, the cell sample is as pure as
possible to prevent reaction between CTL-Ps not directed
against tumor cells and their targets. This is not always
possible, however, and thus it is preferred to treat the
mixture to eliminate natural killer ("NK") like cells
contained therein. The term "natural killer like" includes
natural killer cells, as well as cells which function in the
same or an equivalent manner. One approach to doing this is
by adding, e.q., an NK inhibitor.
The lytic determination can be accomplished by means well
known in the art, including the 5lCr release method, described
~5 supra.
Desirably, when culturing the mixtures described herein,
this is done without the use of feeder cells, however, this is
not a requirement.
The ability to monitor CTL-Ps also enables one to monitor
responses such as the immune response of a subject with
respect to the subject's tumor. ~Changes in CTL-P levels are
indicative of changes in the immune response, and serve an
important diagnostic function in that changes over time,can
indicate a worsening or improvement in the subject's health.
Various modifications to the method described herein have
been shown. Others will be clear to the skilled artisan and
are not repeated here.
The terms and expressions 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 expressions 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.
