Generating MUC1 CTL in Mice 455
455
37
Generation of MUC1 Cytotoxic T-Cells in Mice
and Epitope Mapping
Vasso Apostolopoulos, Ian F. C. McKenzie,
and Geoffrey A. Pietersz
1. Introduction
A successful vaccine for cancer immunotherapy, particularly for solid tumors,
would require a suitable target antigen and the production of a cytotoxic T-cell
response (1). In the mid- to late-1980s, there was a focus on monoclonal antibodies
(MAbs) for the treatment of common cancers, such as those of the colon, breast, and
lung. However, with the difficulties of using such agents, there is now a clear focus on
cellular immunity for several reasons. First, using genetic engineering techniques,
peptide epitopes have been identified and can be produced in large amounts, particu-
larly as synthetic peptides and as recombinant molecules. Second, the description of
many cytokines, combined with the knowledge of antigen processing and presentation
by class I and class II pathways, has led to a degree of sophistication and knowledge in
how to immunize to produce the desired response. These developmets are proving
useful in the generation of new and improved vaccines and the future holds much
promise for the production of effective vaccines to prevent, control, and possibly eradi-
cate many diseases, including cancer. It is now theoretically possible to induce either
antibodies or CTLs to defined polypeptides. However, it remains to be determined
which will be the most effective.
As it will be apparent, MUC1 peptides bind to Class I molecules with variable
affinity, some peptides containing appropriate anchors that bind with high affinity
whereas others contain no known anchors but still bind and induce high-avidity CTLs
(2,3). Furthermore, MUC1 is an interesting molecule and there is good evidence, to be
presented herein, that it is presented by Class I molecules in an unusual manner so that
the peptides are exposed to anti-MUC1 antibodies—being the only peptide to be
described thus far that has this property (3a).

In breast and other cancers in which mucin is expressed, MUC1 appears to be a
useful target for immunotherapy. As has been described extensively, there is an
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
456 Apostolopoulos et al.
upregulation (~100-fold) in MUC1 production in cancer. Such material can clearly be
demonstrated by antibody on the surface of cells, but of greater relevance for this
chapter is that large amounts of material produced intracellularly could lead to presen-
tation of peptides by the class I pathway, as has been described for the endogenous
pathway of antigen presentation. Thus, MUC1 could be a suitable target for CTLs.
The first descriptions of cellular immunity to MUC1 were unusual, in that non-
major histocompatibility complex (MHC) restricted cytotoxic cells were described
(4,5). These studies were important because they showed that patients with cancer had
CTL precursors in their lymph nodes, and it was an enticing suggestion to consider
that stimulation of patients by peptides could activate these cells for successful immu-
notherapy. These findings were provocative and stimulating and led us and others to
commence trials with immunotherapy with MUC1 peptides. Our initial observations
using MUC1 peptides from the variable numbers of tandem repeats (VNTR) (see Chap-
ter 30) led to the production of antibodies in mice and to little cellular immunity (6).
Nonetheless, a clinical trial resulted, which was the first to use MUC1 peptides in a
phase I study, and demonstrated that immune responses were weak or nonexistent;
there were no tumor responses and small MUC1 peptides used alone were not consid-
ered to be satisfactory (7). However, other studies have been used with synthetic
MUC1 peptides in patients in which DTH responses were noted but CTL responses
were poor. We then revised our immunisation procedure to find a strategy where
MUC1 was coupled to mannan to target the mannose receptor (under oxidizing condi-
tions), leading to the satisfactory production of CTLs to MUC1. The strategy was
based on a firm foundation: we had the knowledge that mice that had rejected tumors
expressing human MUC1 could produce CTLs. The aim of the study was therefore to

use synthetic MUC1 peptides or fusion proteins to induce CTLs. Our mode of immu-
nization with mannan was the first to describe the production of CTLs in any strain of
mice to human MUC1. Subsequently, other modes of immunization, particularly with
DNA or delivered by vaccinia, have also led to the production of CTLs. Here, we
describe the induction of CTLs in mice to human MUC1, the definition of epitopes
detected, and how the studies were extended to use HLA-A*0201 transgenic mice in
which further epitopes were also defined.
2. Materials
1. Chemicals: Mannan, ethylene glycol, glutathione (reduced form), glutathione-agarose, isopro-
pyl-β-
D
-thiogalactopyranoside (IPTG), phytohemagglutinin (PHA), and ethanediol were from
Sigma, St. Louis, MO). Sodium periodate, Triton X-100, and glutaraldehyde were from BDH,
Poole, Dorset, UK. PD-10 columns were from Pharmacia, Uppsala, Sweden.
51
Cr-sodium chro-
mate was from Amersham, Amersham, CA. Ampicillin was were from Boehringer Mannheim,
CA. DME, RPMI, penicillin, streptomycin, and fetal calf serum (FCS) were from Common-
wealth, Melbourne, Australia. Bacto-tryptone and bacto-yeast were from Difco, Detroit, MI.
2. Culture media:
a. Growth medium: DME or RPMI supplemented with 10% FCS, 2mM glutamine, 100
IU/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol.
b. Bacterial growth medium: Luria broth (LB) was prepared by mixing bacto-tryptone
(10 g), bacto–yeast extract (5 g), and sodium chloride (10 g) in 950 mL of deionized
Generating MUC1 CTL in Mice 457
water and adjusting the pH to 7.0 with 5 M sodium hydroxide. The volume was
adjusted to 1 L and sterilized by autoclaving for 20 min at 15 lb/in.
2
on liquid cycle.
3. Cell lines: Tumor target cells used were: the mastocytoma cell line P815 (DBA/2 strain

origin, H-2
d
); MUC1 transfected P815 cells (containing the cDNA of the membrane-
anchored form of MUC1) (8), RMA (C57BL/6 strain origin, H-2
b
thymoma) cells, and
the C57BL/6 TAP-deficient cell line RMA-S pulsed with peptide. L-cells transfected with
K
b
, D
b
, D
d
, or L
d
with or without peptide pulsing can also be used as target cells. Human
Epstein-Barr virus (EBV)-transformed B-cells, human breast cancer cell lines (MCF-7
[HLA-A*0201
+
, MUC1
+
], BT-20 [HLA-A*0201
–
, MUC1
+
]), or melanoma cell line
(MF272 [HLA-A*0201
+
, MUC1
–

]). All cell lines and lymphoblasts are grown at 37°C,
10% CO
2
in RPMI, supplemented with 10% FCS, 2 mM glutamine, 100 IU/mL of peni-
cillin, and 100 µg/mL of streptomycin.
4. Buffers and solutions:
a. Phosphate buffered saline (PBS): 2.85 g of disodium hydrogen phosphate dihydrate,
0.624 g of sodium dihydrogen phosphate dihydrate, and 8.766 g of sodium chloride
were dissolved in distilled water and the pH adjusted to 7.2 and volume to 1 L.
b. Bicarbonate buffer: 20 mL of a 0.2 M solution of sodium carbonate and 230 mL of
sodium bicarbonate were mixed and the volume adjusted to 1 L.
c. Phosphate buffer: 0.1 M sodium dihydrogen phosphate titrated with concentrated
sodium hydroxide to pH 6.0.
5. Synthetic peptides: Peptides containing two VNTRs (Cp13-32) were synthesized using
an Applied Biosystems Model 430A automated peptide synthesizer. Overlapping 9-mer
peptides spanning the MUC1 VNTR with single amino acid sequence changes were syn-
thesized by Chiron Mimotopes, Victoria, Australia.
6. Inbred mice: Balb/c, C57BL/6, CBA; recombinant mice: H-2K
k
D
b
B10.A(2R), H-2K
b
D
d
,
B10.A(5R). HLA-A*0201/K
b
(9) mice were obtained from the Scripps Clinic and
Research Foundation, California. All mice were 8 to 10 wk old.

3. Methods
3.1. Production of Soluble MUC1 Fusion Protein
1. Grow Escherichia coli transformed with pGEX-3X plasmids containing coding region of five
repeats of the MUC1 VNTR (10–12) overnight in LB containing ampicillin (100 µg/mL) .
2. Dilute the culture 1:25 with fresh (LB) medium and grow bacteria for a further 1 h at 37°C.
3. Add 0.1 mM IPTG to induce the production of recombinant protein and incubate a further
3 h at 37°C.
4. Centrifuge at 2,500g for 15 min at 4°C and resuspend the pellet in 1:10 culture volume of
PBS. Perform all subsequent steps at 4°C (see Note 1).
5. Lyse cells by sonication (3 × 30 s) and add 1% Triton X-100.
6. Centrifuge cells at 10,000g for 15 min at 4°C.
7. Mix the supernatant containing the soluble fusion protein with a 50% solution of glu-
tathione-agarose beads (supernatant:agarose beads, 5:1) on a rotating platform.
8. Collect the beads by centrifugation at 500g for 5 min, and wash three times with PBS.
9. Elute the fusion protein with free glutathione using three 5-min washes with 1.5 bead
volume of 50 mM Tris-HCl (pH 8.0) containing 5 mM reduced glutathione (see Note 2).
10. Dialyze the supernatants into PBS.
11. Measure the optical density at 280 nm, and calculate the concentration using the follow-
ing formula:concentration (mg/mL) = (OD
280
× 10)/14.3.
12. Store at –20°C in aliquots.
458 Apostolopoulos et al.
3.2. Conjugation of MUC1 Fusion Protein or Peptides to Mannan
1. Disssolve 14 mg of mannan in 1 mL of 0.1 M phosphate buffer and leave on ice (13).
2. Make a 0.1 M solution of sodium periodate and add 100 µL to the mannan and leave on
ice for 1 h.
3. Stop the oxidation by adding 10 mL of ethylene glycol and leave on ice for a further 30 min.
4. Pass the reaction mixture through a PD-10 column (void volume = 2.5 mL) and collect 2
mL of oxidized mannan fraction.

5. Add 0.7 mg of MUC1 FP to the oxidized mannan and react overnight at room temperature
to form the conjugate (MFP) (see Note 3).
6. Store the MFP in aliquots at -20
o
C.
3.3. Mice and Immunization Schedule (14)
1. Immunize mice intraperitoneally with 5 µg of MFP (5 µg = to the amount of FP) or the
same amount of peptides (linked to keyhole limpet hemocyanin [KLH]) weekly for 3 wk
(see Note 4).
3.4. Preparation of Target Cells
1. Prepare lymphoblast target cells by placing 2 × 10
6
spleen cells in wells of a 24-
well plate with 1 µg/mL of PHA-L, and incubate for 48 h at 37˚C in 10% CO
2
to
form blasts cells.
2. Incubate blast cells overnight with 20 µM peptide (either 20 mer or short 9 mers for
epitope mapping).
3. Use the tumor target cells, P815, P815 transfected with the MUC1 cDNA, RMA, single
H-2 alleles expressed after L-cell transfection, and TAP-deficient RMA-S cells (used as
CTL targets and in stabilization assays). For HLA-A*0201 mice, use the following target
cells: PHA blasts from autologous mice, human EBV transformed B cells, PHA pulsed
human peripheral blood mononuclear cells (PBMC), human breast cancer cell lines,
(MCF-7 [HLA-A*0201
+
, MUC1
+
]), BT-20 (HLA-A*0201
–

, MUC1
+
) or melanoma cell
line (MF272 [HLA-A*0201
+
, MUC1
–
]).
4. Radiolabel 10
6
peptide-pulsed blast cells or 10
6
tumor target cells with 100 µCi of
Na
2
51
CrO
4
(
51
Cr) for 60 min at 37°C.
3.5. Cytotoxic T-Lymphocyte Assay
1. Immunize mice with MFP.
2. Sacrifice mice 7–10 d after the final injection, collect their spleen cells, remove red cells,
and wash with 2% FCS/PBS.
3. Radiolabel 10
6
target cells with 100 µCi of Na
2
51

CrO4 (Amersham) for 60 min at 37°C.
4. Resuspend spleen cells and target cells, in culture medium, and then combine in various
effector-to-target ratios (100:1 to 5:1) in triplicate, in 96-well U-bottomed plates.
5. To ascertain spontaneous release (medium alone) and maximum release (after treat-
ment with 10% sodium dodecyl sulfate [SDS]) set up six wells with labeled target
cells alone.
6. Centrifuge the plates at 100g for 3 min to initiate cell contact, and incubate for 4 h at 37°C
in 10% CO
2
.
7. After incubation, centrifuge again, collect supernatants, and quantitate radioactivity in a
gamma counter.
8. Determine specific
51
Cr release as follows: [(experimental – spontaneous)/(maximum –
spontaneous)] × 100%.
Generating MUC1 CTL in Mice 459
3.6. Cytotoxic T-Lymphocyte Precursor (CTLp) Assay
1. Ten to 14 d after the last immunization, sacrifice mice and collect effector spleen cells.
2. Set up 32 replicates for at least six effector cell numbers (1 × 10
3
–1.28 × 10
5
) in
U-bottomed microtiter trays together with 5 × 10
5
mitomycin C-treated spleen cells (or
spleen cells irradiated with 50,000 rad) in RPMI supplemented with 10% FCS, antibiot-
ics, 5 µM VNTR peptide, and 10 U/mL of rhIL-2.
3. Set up control wells containing stimulator cells alone, with peptide, and with IL-2 only.

4. Seven days later replace 100 µL of supernatant with 100 µL of target cell suspension
containing 10
451
Cr-labeled targets.
5. After 4 h quantitate the radioactivity in a gamma counter.
6. Calculate the mean ± standard deviation (SD) of specific
51
Cr release from the controls.
7. Calculate the fraction of wells with cytolytic activity by counting the number of wells
from 32 of each effector cell number with radioactivity greater than mean + 3 SDs.
8. Graph the fraction of negative wells on a logarithmic scale (y-axis) vs the effector cell
number (x-axis).
9. Determine the CTLp frequency from the graph as the responder cell dose required to
generate 37% negative wells (see Note 5 and Fig. 1).
Fig. 1. Typical example of a graph depicting the CTLp frequency (10,000) for MFP immu-
nized mice. (············), 0.37% negative wells; (———), CTLp frequency.