Glycoprotein Methods and Protocols - P34

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Generating MUC1 CTL in Mice 45545537Generation of MUC1 Cytotoxic T-Cells in Miceand Epitope MappingVasso Apostolopoulos, Ian F. C. McKenzie,and Geoffrey A. Pietersz1. IntroductionA successful vaccine for cancer immunotherapy, particularly for solid tumors,would require a suitable target antigen and the production of a cytotoxic T-cellresponse (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, andlung. However, with the difficulties of using such agents, there is now a clear focus oncellular 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 ofmany cytokines, combined with the knowledge of antigen processing and presentationby class I and class II pathways, has led to a degree of sophistication and knowledge inhow to immunize to produce the desired response. These developmets are provinguseful in the generation of new and improved vaccines and the future holds muchpromise for the production of effective vaccines to prevent, control, and possibly eradi-cate many diseases, including cancer. It is now theoretically possible to induce eitherantibodies or CTLs to defined polypeptides. However, it remains to be determinedwhich will be the most effective.As it will be apparent, MUC1 peptides bind to Class I molecules with variableaffinity, some peptides containing appropriate anchors that bind with high affinitywhereas 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 bepresented herein, that it is presented by Class I molecules in an unusual manner so thatthe peptides are exposed to anti-MUC1 antibodies—being the only peptide to bedescribed thus far that has this property (3a).In breast and other cancers in which mucin is expressed, MUC1 appears to be auseful target for immunotherapy. As has been described extensively, there is anFrom:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 456 Apostolopoulos et al.upregulation (~100-fold) in MUC1 production in cancer. Such material can clearly bedemonstrated by antibody on the surface of cells, but of greater relevance for thischapter 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 endogenouspathway 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 hadCTL precursors in their lymph nodes, and it was an enticing suggestion to considerthat stimulation of patients by peptides could activate these cells for successful immu-notherapy. These findings were provocative and stimulating and led us and others tocommence trials with immunotherapy with MUC1 peptides. Our initial observationsusing 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 aphase 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 syntheticMUC1 peptides in patients in which DTH responses were noted but CTL responseswere poor. We then revised our immunisation procedure to find a strategy whereMUC1 was coupled to mannan to target the mannose receptor (under oxidizing condi-tions), leading to the satisfactory production of CTLs to MUC1. The strategy wasbased on a firm foundation: we had the knowledge that mice that had rejected tumorsexpressing human MUC1 could produce CTLs. The aim of the study was therefore touse 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 ofmice to human MUC1. Subsequently, other modes of immunization, particularly withDNA or delivered by vaccinia, have also led to the production of CTLs. Here, wedescribe the induction of CTLs in mice to human MUC1, the definition of epitopesdetected, and how the studies were extended to use HLA-A*0201 transgenic mice inwhich further epitopes were also defined.2. Materials1. Chemicals: Mannan, ethylene glycol, glutathione (reduced form), glutathione-agarose, isopro-pyl-β-D-thiogalactopyranoside (IPTG), phytohemagglutinin (PHA), and ethanediol were fromSigma, St. Louis, MO). Sodium periodate, Triton X-100, and glutaraldehyde were from BDH,Poole, Dorset, UK. PD-10 columns were from Pharmacia, Uppsala, Sweden. 51Cr-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, 100IU/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 457water and adjusting the pH to 7.0 with 5 M sodium hydroxide. The volume wasadjusted 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 strainorigin, H-2d); MUC1 transfected P815 cells (containing the cDNA of the membrane-anchored form of MUC1) (8), RMA (C57BL/6 strain origin, H-2bthymoma) cells, andthe C57BL/6 TAP-deficient cell line RMA-S pulsed with peptide. L-cells transfected withKb, Db, Dd, or Ldwith or without peptide pulsing can also be used as target cells. HumanEpstein-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% CO2in 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 chloridewere 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 ofsodium bicarbonate were mixed and the volume adjusted to 1 L.c. Phosphate buffer: 0.1 M sodium dihydrogen phosphate titrated with concentratedsodium hydroxide to pH 6.0.5. Synthetic peptides: Peptides containing two VNTRs (Cp13-32) were synthesized usingan Applied Biosystems Model 430A automated peptide synthesizer. Overlapping 9-merpeptides 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-2KkDbB10.A(2R), H-2KbDd,B10.A(5R). HLA-A*0201/Kb(9) mice were obtained from the Scripps Clinic andResearch Foundation, California. All mice were 8 to 10 wk old.3. Methods3.1. Production of Soluble MUC1 Fusion Protein1. Grow Escherichia coli transformed with pGEX-3X plasmids containing coding region of fiverepeats 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 further3 h at 37°C.4. Centrifuge at 2,500g for 15 min at 4°C and resuspend the pellet in 1:10 culture volume ofPBS. 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 beadvolume 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) = (OD280× 10)/14.3.12. Store at –20°C in aliquots. 458 Apostolopoulos et al.3.2. Conjugation of MUC1 Fusion Protein or Peptides to Mannan1. 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 onice 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 2mL of oxidized mannan fraction.5. Add 0.7 mg of MUC1 FP to the oxidized mannan and react overnight at room temperatureto form the conjugate (MFP) (see Note 3).6. Store the MFP in aliquots at -20oC.3.3. Mice and Immunization Schedule (14)1. Immunize mice intraperitoneally with 5 µg of MFP (5 µg = to the amount of FP) or thesame amount of peptides (linked to keyhole limpet hemocyanin [KLH]) weekly for 3 wk(see Note 4).3.4. Preparation of Target Cells1. Prepare lymphoblast target cells by placing 2 × 106spleen 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% CO2toform blasts cells.2. Incubate blast cells overnight with 20 µM peptide (either 20 mer or short 9 mers forepitope mapping).3. Use the tumor target cells, P815, P815 transfected with the MUC1 cDNA, RMA, singleH-2 alleles expressed after L-cell transfection, and TAP-deficient RMA-S cells (used asCTL targets and in stabilization assays). For HLA-A*0201 mice, use the following targetcells: PHA blasts from autologous mice, human EBV transformed B cells, PHA pulsedhuman peripheral blood mononuclear cells (PBMC), human breast cancer cell lines,(MCF-7 [HLA-A*0201+, MUC1+]), BT-20 (HLA-A*0201–, MUC1+) or melanoma cellline (MF272 [HLA-A*0201+, MUC1–]).4. Radiolabel 106peptide-pulsed blast cells or 106tumor target cells with 100 µCi ofNa251CrO4 (51Cr) for 60 min at 37°C.3.5. Cytotoxic T-Lymphocyte Assay1. 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 106target cells with 100 µCi of Na251CrO4 (Amersham) for 60 min at 37°C.4. Resuspend spleen cells and target cells, in culture medium, and then combine in variouseffector-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 targetcells alone.6. Centrifuge the plates at 100g for 3 min to initiate cell contact, and incubate for 4 h at 37°Cin 10% CO2.7. After incubation, centrifuge again, collect supernatants, and quantitate radioactivity in agamma counter.8. Determine specific 51Cr release as follows: [(experimental – spontaneous)/(maximum –spontaneous)] × 100%. Generating MUC1 CTL in Mice 4593.6. Cytotoxic T-Lymphocyte Precursor (CTLp) Assay1. 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 × 103–1.28 × 105) inU-bottomed microtiter trays together with 5 × 105mitomycin C-treated spleen cells (orspleen 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 suspensioncontaining 10451Cr-labeled targets.5. After 4 h quantitate the radioactivity in a gamma counter.6. Calculate the mean ± standard deviation (SD) of specific 51Cr release from the controls.7. Calculate the fraction of wells with cytolytic activity by counting the number of wellsfrom 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 cellnumber (x-axis).9. Determine the CTLp frequency from the graph as the responder cell dose required togenerate 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. 460 Apostolopoulos et al.3.7. Epitope Mapping (3,15,16) (see Note 6)1. For generation of CTL, see Subheading 3.3.1.2. For generation of targets, Subheading 3.4.3. Peptide pulsing: Incubate target cells overnight with 20 µM peptide and label with 51Cr asdescribed under Subheading 3.4. (see Note 7).4. Set up CTL assays as under Subheading 3.5., item 4 at a constant effector-to-target ratio.5. Graph the 9-mer sequence (y-axis) and specific lysis (x-axis) as a horizontal bar graph todetermine the epitope (Fig. 2)6. Examples of epitope mapping: Effector spleen cells from C57BL/6 (H2KbDb) mice immunizedwith M-FP are mixed with B10.A(5R) (H2KbDd) peptide pulsed lymphoblast target cells sothat only the reaction with H2Kbis determined. Similarly, H2Dbis mapped using recombinantmice, with L-cells transfected with DbcDNA, and specificity is confirmed in RMA-S cells.H2d is mapped similarly. For HLA-A*0201 epitope mapping, EBV-immortalized B-cells(HLA-A*0201, HLA-A1, HLA-A11, and so forth) and PHA lymphoblast target cells derivedfrom HLA-A*0201/Kb mouse spleen cells or from human PBMC are used.4. Notes1. The pellet may be stored at –70°C for a couple of months before resuspending in PBS.2. As an alternative to batch processing, a chromatography column packed with GST-agar-ose beads may be used. The supernatant can be recirculated through the column, washedFig. 2. Typical example of a graph showing the mapping of the Kbepitope (SAPDTRPAP)presented by MUC1. CTL assay using C57BL/6 (H2b) effectors and B10.A(5R) (KbDd) PHA blasts. Generating MUC1 CTL in Mice 461and eluted with GSH using a fast-performance liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC).3. Peptides were linked to mannan via KLH. Peptides were linked to KLH using glutaralde-hyde. Briefly, dissolve the peptide (2 mg) in 1.75 mL of PBS and mix with 0.25 mL ofKLH (2 mg/mL). Add 0.25% glutaraldehyde (1 mL) and mix overnight at room tempera-ture in the dark. Dialyze the mixture overnight into PBS with several changes. Add thedialyzed mixture to 1 mL of oxidized mannan.4. CTL can readily be generated in any strain of mice and measured directly. We haveimmunized 15 strains and without fail obtain 60% lysis at an effector-to-target ratio of20:1. Three immunizations are optimal—more or less gives less lysis. As an alternative tothree immunizations by the IP route, one injection of in vitro sensitized macrophages/dendritic cells will suffice. For this, adherent cells are exposed to MFP overnight, washed,and then injected. The CTLp frequency (see Note 5) is ~1/10,000—the same as obtainedwith three IP injections.5. The CTLp frequency obtained depends on the number of immunizations performed: threeinjections gives ~1/10,000 or greater, one injection gives ~1/80,000, and two injectionsgives ~1/30,000. It appears that a frequency of >1/20,000 is required to cause tumorrejection.6. Mucin 1 peptides are processed by different cells and act as antigen-presenting cells;we have used adherent cells, PHA blasts, or EBV-infected cells. Peptides 30–105 merare processed and presented as 8 to 9 mers. Alternatively, small peptides (8 to 9 mers)can be surface loaded on to cells.7. Epitopes are mapped by preparing direct CTL on peptide-loaded/pulsed target cells anddetermining which peptides lead to lysis.References1. Singer, A. (1994) News: Time and truth for cancer vaccines - a new generation. J. Natl.Cancer Inst. 86, 330–330.2. Dahl, A. M., Beverley, P. C., and Stauss, H. J. (1996) A synthetic peptide derived from thetumor-associated protein mdm2 can stimulate autoreactive, high avidity cytotoxic T lym-phocytes that recognize naturally processed protein. J. Immunol. 157, 239–246.3. Apostolopoulos, V., Haurum, J. S., and McKenzie, I. F. C. (1997) MUC1 peptide epitopesassociated with 5 different H2 Class I molecules. Eur. J. Immunol. 27, 2579–2587.3a. Apostolopoulos, V., Chelvanayagam, G., Xing, P. X., and McKenzie, I. F. L. (1998) Anti-MUC1 antibodies react directly with MUC1 peptides presented by Class1 H2 and HLAmolecules. J. Immunol. 161, 767–775.4. Barnd, D. L., Lan, M. S., Metzgar, R. S., and Finn, O. J. (1989) Specific major histocom-patibility complex-unrestricted recognition of tumor associated mucins by human cyto-toxic T cells. Proc. Natl. Acad. Sci. USA 86, 7159–7163.5. Ioannides, C. G., Fisk, B., Jerome, K. R., Irimura, T., Wharton, J. T., and Finn, O. J.(1993) Cytotoxic T cells from ovarian malignant tumors can recognize polymorphic epi-thelial mucin core peptides. J. Immunol. 151, 3693–3703.6. Apostolopoulos, V., Xing, P. X., and McKenzie, I. F. C. (1994) Murine immune responseto cells transfected with human MUC1: immunisation with cellular and synthetic antigens.Cancer Res. 54, 5186–5193.7. Xing, P. 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(1988) Single-step purification of polypeptides expressedin E. coli as fusions with glutathione S-transferase. Gene 67, 31–40.12. Apostolopoulos, V., Xing, P. X., Trapani, J. A., and McKenzie, I. F. C. (1993) Productionof anti-breast cancer monoclonal antibodies using a glutathione-S-transferase-MUC1 bac-terial fusion protein. Br. J. Cancer 67, 713–720.13. Apostolopoulos, V., Pietersz, G. A., and McKenzie, I. F. C. (1996) Cell-mediated immuneresponses to MUC1 fusion protein coupled to mannan. Vaccine 14, 930–938.14. Pietersz, G. A., Wenjun, L., Popovski, V., Caruana, J.-A., Apostolopoulos, V., andMcKenzie, I. F. C. (1998) Parameters in using mannan-fusion protein (MFP) to inducecellular immunity. Cancer Immunol. Immunother., 45, 321–326.15. Apostolopoulos, V., Loveland, B. E., Pietersz, G. A., and McKenzie, I. F. C. (1995) CTL inmice immunised with human Mucin 1 are MHC-restricted. J. Immunol. 155, 5089–5094.16. Apostolopoulos, V., Karanikas, V., Haurum, J., and McKenzie, I. F. C. (1997) Inductionof HLA-A2 restricted cytotoxic T lymphocytes to the MUC1 human breast cancer antigen.J. Immunol. 159, 5211–5218. . target cells. HumanEpstein-Barr virus (EBV)-transformed B-cells, human breast cancer cell lines (MCF-7[HLA-A*0201+, MUC1+], BT-20 [HLA-A*0201–, MUC1+]), or. RMA, singleH-2 alleles expressed after L-cell transfection, and TAP-deficient RMA-S cells (used asCTL targets and in stabilization assays). For HLA-A*0201