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[Cancer Research 64, 8077-8084, November 1, 2004]
© 2004 American Association for Cancer Research

Immunology

A Unique Mucin Immunoenhancing Peptide with Antitumor Properties

Lynn M. Herbert1, Joseph F. Grosso1, Mantley Dorsey, Jr.1, Tihui Fu1, Iafa Keydar2, Mabel A. Cejas1, Daniel H. Wreschner2, Nechama Smorodinski2 and Diana M. Lopez1

1 Department of Microbiology & Immunology, University of Miami School of Medicine, Miami, Florida; and 2 Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucin 1 (MUC1), a polymorphic type 1 transmembrane protein, which has a large, heavily glycosylated extracellular domain, a transmembrane domain, and a cytoplasmic tail, is found on the apical surface of normal cells lining the lumen of ducts and glands (1 , 2) , normal epithelium (3) , lymphocytes (4 , 5) , and dendritic cells (6 , 7) . The extracellular domain polymorphism is derived from the 20 amino acid repeat unit, which can vary from 20 to 100 repeats and has five potential O-linked glycosylation sites per repeat. MUC1 has been selected as a mediator of cell-cell interactions because of its proposed dual role as both an adhesive and antiadhesive molecule (8) and in signaling via the cytoplasmic tail (9 , 10) .

Breast tumors express very high levels of an altered form of the MUC1 glycoprotein (11) as do several other types of human cancers, including those of the pancreas, uterus, and colon (1) , and this hypoglycosylated form of MUC1 is overexpressed on the entire surface of the transformed cells. The alteration of MUC1 in neoplasia makes it an ideal tumor antigen for use both diagnostically and in therapy, and several studies highlight the potential of the MUC1 molecule in the development of tumor-antigen–specific vaccines (12, 13, 14) .

A secreted isoform of MUC1, MUC1/sec, was initially isolated from an infiltrating ductal breast carcinoma (15) and subsequently also found in patients with ovarian and cervical cancers. MUC1/sec is an alternative splice variant of the MUC1 gene, which includes the first 33 bp of intron 2, followed by a stop codon, and the protein therefore lacks the cytoplasmic and transmembrane domains. However, the extracellular portion of MUC1/sec, like that of MUC1, includes varying numbers of the tandem repeat sequences. Because MUC1/sec lacks both the transmembrane and cytoplasmic domains, it is secreted from the cells. Additionally, MUC1/sec contains an 11 amino acid peptide at its COOH terminus that is not found in other mucins.

To study immune responses against mammary tumors with a defined tumor antigen, we transfected DA-3 cells, a murine mammary tumor cell line developed in our laboratory, with the human transmembrane MUC1 isoform, DA-3/TM, the human secreted form, DA-3/sec, or the neomycin vector alone, DA-3/neo. Both the DA-3/TM and DA-3/neo cells produced viable tumors in intact BALB/c mice. However, the transfected DA-3/sec cells did not produce viable tumors in intact BALB/c animals but did grow in nude BALB/c animals. Furthermore, previous exposure to DA-3/sec cells conferred protection against challenge with the DA-3/TM or DA-3/neo cells and two other nonrelated tumors, a renal carcinoma and an osteosarcoma. The studies presented herein also indicate that the unique 11 amino acid peptide present in the secreted MUC1 isoform appears to be involved in the observed phenomenon and may serve as an immunoenhancing molecule.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Tumors.
BALB/c mice used in these studies were 8 to 12 weeks of age and bred and used in our animal facility at the University of Miami according to the guidelines of the NIH. BALB/c nu+/nu+ were purchased from Taconic Laboratory (Germantown, NY). The monoclonal DA-3 mammary tumor cell line, derived in our laboratory from the D1-DMBA-3–transplantable mammary adenocarcinoma, and the monoclonal transfectants were maintained as published previously (16) . The renal carcinoma (RENCA) cells were maintained in RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, 1% sodium pyruvate, 1% nonessential amino acids, and 50 mmol/L 2-mercaptoethanol. The K-7 osteosarcoma cells were grown in DMEM/high glucose with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin. The RENCA and K-7 cell lines were a gift from Dr. Eduardo Sotomayor (Moffit Cancer Center, Tampa, FL). Lewis lung carcinoma cells, obtained from the American Type Culture Collection (Manassas, VA), were maintained per recommended conditions. Tumors were implanted by s.c. injection of 1 x 106 tumor cells, resulting in measurable tumors 7 to 10 days after implantation.

Transfection of DA-3 Cells.
Stable transfectants expressing the various MUC1 isoforms were generated by transfecting an expression plasmid (pSV2 neo) harboring either MUC1/TM or MUC1/sec DNA into DA-3 mouse mammary tumor cells as described previously (17) . Control cells were transfected with the vector containing only the neomycin resistance gene.

Peptide Fragment and Antibody Preparation.
The H23 monoclonal antibody (18) was used to detect the tandem repeat sequence common to both MUC1/TM and MUC1/sec. Antibody 1709, which recognizes only the secreted form of MUC1, was prepared by immunizing chickens with the MUC1/sec-specific peptide VSIGLSFPMLP conjugated to malaimide-activated keyhole limpet hemocyanin (KLH). Free peptide concentrations and conjugations of at least 150 peptide molecules per KLH molecule were used for immunization. The MUC1/sec-specific peptide was prepared either conjugated to KLH or as a multiple antigenic peptide by Aves Laboratory (Tigand, OR). Synthesis of the scrambled version of the MUC1/sec peptide (VFGPSLMPLIS) in the context of a multiple antigenic peptide was also done by Aves Laboratory.

RNA Analysis.
For the reverse transcription-PCR analysis, total RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH). One microgram of total RNA per sample was reverse transcribed with a Primus thermocycler (MWG Biotech, High Point, NC) and the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA) with oligo d(T)16 primers, according to the manufacturers’ instructions. The reaction was incubated for 60 minutes at 42°C, followed by inactivation of the murine leukemia virus reverse transcriptase at 99°C for 5 minutes. The cDNA products obtained were subjected to PCR amplification for MUC1, MUC/sec, and ß-actin under the following conditions: 94°C for 10 minutes, followed by 35 cycles of 94°C for 45 seconds, 57°C for 45 seconds, 72°C for 30 seconds, with a final extension of 10 minutes at 72°C. The primers were as follows: human MUC1 and human MUC1/sec sense, 5'-TGCATCAGGCTCAGCTTCTA-3'; human MUC1 antisense, 5'-GAAATGGCACATCACTCACG-3'; human MUC1/sec antisense, 5'-GGAAGGAAAGGCCGATACTC-3'; murine ß-actin sense, 5'-TCTGGCACCACACCTTCTAC-3', and murine ß-actin antisense, 5'-GAAGGAAGGCTGGAAGAGTG-3'. Fifteen microliters of the amplified DNA fragments were electrophoresed on a 1.6% agarose gel stained with ethidium bromide and visualized by UV transillumination.

Western Blot Analysis.
Equal amounts of tumor cell supernatants were separated on 6% SDS polyacrylamide gels under reducing conditions and then transferred onto 0.45-µm pore size Protran nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH) with a trans-Blot electrophoretic cell (Bio-Rad, Hercules, CA). Membranes were blocked for 1 hour at room temperature in 5% nonfat dry milk in 1x TBS-0.1% Tween 20, followed by 1 hour of incubation at room temperature with antibody against the tandem repeat portion of human MUC1. Blots were washed for 30 minutes with three changes of 1x TBS-0.1% Tween 20 solution, followed by 1 hour of incubation at room temperature with the horseradish peroxidase-conjugated antirabbit IgG antibody (Chemicon International, Temecula, CA). Blots were washed again for 30 minutes and incubated for 5 minutes with Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The results were visualized by exposing blots to BioMax autoradiographic film (Kodak, Rochester NY).

Cytotoxicity Assays.
Unseparated splenic cells used in the natural killer cell assays were prepared as described previously (19) . Effector cells at a concentration of 25 x 106/mL were added to microtiter plate wells in volumes ranging from 25 to 150 µL, depending on the final effector to target ratio desired. Twenty-five microliters of 51Cr-labeled YAC-1 target cells at 1 x 106 cells/mL were added to all wells so that the final volume in each well was 200 µL. After incubation for 4 hours at 37°C in 5% CO2, 100 µL of medium were removed from each well and counted on a Packard MultiPrias 2 gamma counter. Percent cytotoxicity was calculated according to the following equation: % cytotoxicity = (experimental counts – 0% control)/total counts x 100, when the 0% control represented the amount of 51Cr released in cultures of target cells alone after 4 hours of incubation, and the total counts was determined by counting 100 µL of target cells alone lysed with 2% NP40.

Standard CTL assays were done as described previously (20) . Briefly, 1 x 107 splenic cells from mice that received injections of tumor cells were mixed with 5 x 104 mitomycin C-treated tumor cells (100 µg/mL, 37°C, 60 minutes) and cocultured for 5 days in 2 mL of RPMI 1640 containing 10% FCS, 20 mmol/L HEPES, 100 units/mL penicillin, 100 µg/mL streptomycin, and 5 x 10-5 mol/L 2-mercaptoethanol in a 24-well plate (Corning Labware & Equipment, Corning, NY) at 37°C in 5% CO2 atmosphere. After 5 days, the activated lymphoid cells were harvested, and their cytotoxicity was assessed in a standard 4-hour 51Cr release assay (21) .

T-Cell Subset Purification and Fluorocytometric Analysis.
Microbeads conjugated with monoclonal antibodies to either CD4 or CD8 (Miltenyi Biotec, Inc., Auburn, CA) were used to purify T-cell subsets according to the manufacturer’s directions (22) . The purity of the T-cell subsets was determined by staining the cells with anti-CD4 and anti-CD8 antibodies (BD PharMingen, San Diego, CA) and analyzing them on a BD PharMingen LSR Analyzer.

In vivo Protection Studies.
BALB/c mice were inoculated s.c. twice, 2 weeks apart, with 106 DA-3/sec tumor cells. Two weeks after the second inoculation, the animals were challenged with 106 tumor cells mixed with 106 DA-3/sec tumor cells. Animals were monitored for tumor growth, and the tumor size was recorded. In other studies, we used the peptide synthesized by Aves Laboratory composed of the unique 11 amino acid sequence present in the secreted MUC1 isoform bound to KLH. This KLH-peptide was administered twice before challenge with the various tumor cells as indicated. The equivalent of 50 µg of peptide plus the conjugated KLH was administered with complete Freund’s adjuvant for the first inoculation. Subsequently, at 2-week intervals, 25 µg of the peptide:conjugate in incomplete Freund’s adjuvant was administered. Two weeks after the final inoculation, the mice were challenged with 106 tumor cells mixed with 25 µg of peptide:conjugate and no adjuvant. In all studies, the control group consisted of untreated mice given the challenge tumor cells. Tumor growth was monitored every 2 or 3 days, and tumor size was measured by calipers. In additional studies, mice were pretreated with a multiple antigenic peptide preparation containing either the 11 amino acid peptide (multiple antigenic peptide-immunoenhancing peptide) or a scrambled version of the peptide (multiple antigenic peptide-scrambled) as described above. Multiple antigenic peptide conjugates containing 50 µg of peptide were injected three times at 3-day intervals without Freund’s adjuvant. The challenge was mixed with either multiple antigenic peptide-immunoenhancing peptide or multiple antigenic peptide-scrambled and given 3 days after the last treatment.

Statistical Analysis.
Student’s t test was used for statistical analysis of the experiments. P < 0.05 was used as the level of significance. Kaplan-Meier curves with log-rank analysis were used to determine whether the time to tumor formation was significant for the in vivo protection studies.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of MUC1/sec Prevents Tumor Growth in DA-3 Cells.
After transfection, reverse transcription-PCR was used to confirm the presence of MUC1 on the cells. Fig. 1ACitation shows that DA-3/sec cells contained a band corresponding to the human form of MUC1/sec (Fig. 1ACitation , Lane 2), and DA-3/TM cells express the MUC1-specific sequences (Fig. 1ACitation , Lane 3). Untransfected DA-3 cells do not express any MUC1-related sequences (Fig. 1ACitation , Lanes 4 and 5). Actin controls for each cell type are shown in Fig. 1ACitation , Lanes 6 to 8. In additional studies, the DA-3/neo, DA-3/TM, and DA-3/sec, cells, as well as the parent cell line, were grown in vitro, and after 2 days, the culture supernatants were tested for the presence of MUC1 by Western blot analyses. As seen in Fig. 1BCitation , with the H23 antibody specific for the human MUC1 tandem repeat, only the DA-3/sec cells secreted this mucin (Fig. 1BCitation , Lane 5).



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  		Fig. 1. MUC1/sec-transfected cells remain viable yet fail to grow in vivo. DA-3 cells were transfected with plasmids encoding either the full-length MUC1 gene (DA-3/TM) or the MUC1/sec gene (DA-3/sec). A. Subsequent to antibiotic selection, RNA was isolated to generate cDNA, and PCR was done for the presence of either MUC1 or MUC1/sec message. Lanes 1: molecular weight marker; Lane 2: DA-3/sec cDNA amplified with MUC1/sec primers; Lane 3: DA-3/TM cDNA amplified with MUC1 primers; Lane 4: DA-3 cDNA amplified with MUC1/sec primers; Lane 5: DA-3 cDNA amplified with MUC1 primers; Lanes 6–8: DA-3/sec, DA-3/TM, and DA-3 cDNA amplified with ß-actin primers. B. One hundred micrograms of total protein from supernatants from tumor cell lines grown 3 days in culture were analyzed by Western blot analysis with the H23 antibody specific for MUC1 tandem repeats. Only DA-3/sec cell supernatant is positive for H23 staining, consistent with MUC1/sec secretion. Lanes 1: HeLa, Lanes 2: CTLL2, Lanes 3: DA-3, Lanes 4: DA-3/TM, Lanes 5: DA-3/sec, 6: DA-3/neo, Lanes 7: HeLa, and Lanes 8: DA-3/sec. Lanes 7 and 8 incubated with secondary antibodies only. C. A total of 1 x 106 DA-3, DA-3/neo, DA-3/TM, or DA-3/sec cells was s.c. injected into 8 to 12-week old BALB/c mice to assess their ability to form tumors in vivo. Animals were monitored for survival over a several month period. Mice receiving DA-3/sec cells remained healthy over the course of the entire experiment. D, in vitro growth kinetics of DA-3 cells and transfectants. Cells were seeded in 6-well tissue culture plates at an initial density of 2 x 106. At 24, 48, 72, and 96 hours after initial seeding, the appropriate wells were trypsinized and counted using trypan blue to exclude dying cells. DA-3 cells transfected with MUC1/sec have faster in vitro growth kinetics than the other cell types.

 
All four DA-3 cell lines were used for in vivo experiments to determine tumor incidence and time of appearance in BALB/c mice. Implantation of DA-3, DA-3/neo, and DA-3/TM mammary tumor cells into mice gave rise to palpable tumors of approximately equal size by 7 days, and by 15 days, essentially all animals had measurable tumors (Table 1)Citation . In sharp contrast, the DA-3 cells transfected with the secreted isoform of MUC1, DA-3/sec failed to produce viable tumors. Remarkably, these animals remained tumor free > 12 months after implantation, whereas animals with the other three DA-3 cell types failed to survive past 100 days (Fig. 1C)Citation . In other studies, mice implanted with 1, 2, or 10 x 106 cells DA-3/sec cells showed no tumor growth even at the highest tumor cell concentration. Inoculation of 20 x 106 DA-3/sec cells resulted in initial tumors at 3 days, which began to regress at day 7, and completely disappeared by day 10 (data not shown).


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  		Table 1 Incidence and time of appearance of DA-3, DA-3/neo, DA-3/TM or DA-3/sec tumors in BALB/c mice

 
One explanation for these results was that the transfection process selectively impaired the basic growth potential of the DA-3/sec cells. However, the in vitro growth kinetics of the four DA-3 tumor cell types were similar, indicating that the transfection process did not alter their growth potential (Fig. 1D)Citation .

Immunocompromised Mice Support the Growth of DA-3/sec Cells.
To determine whether the DA-3/sec cells had lost in vivo tumorigenic potential, the four DA-3 cell lines were implanted in BALB/c nu+/nu+ mice, and tumor incidence and size were recorded. Fig. 2ACitation shows that the DA-3, DA-3/neo, and DA-3/TM cells caused tumors by 7 days after implantation in nude BALB/c mice. In contrast to the results shown for intact BALB/c in Table 1Citation and Fig. 1CCitation , 30% of the nude mice that received DA-3/sec tumor cells developed tumors by 14 days after inoculation, and by day 25, all of the nude animals had tumors (Fig. 2A)Citation . Thus, the failure of DA-3/sec cells to produce tumors in the intact BALB/c mice appears to be immunologically controlled because implantation of this tumor in nude mice did result in 100% tumor development, albeit with altered kinetics. To confirm these findings, two additional DA-3/sec cell transfectants were implanted into both nude and intact BALB/c animals. Although none of the DA-3/sec transfectants grew in the immunocompetent BALB/c mice, all three, i.e., DA-3/sec, DA-3/sec 11, and DA-3/sec 22, grew in BALB/c nude mice, although the growth kinetics of the three cell lines were somewhat different (Fig. 2B)Citation . Interestingly, implantation of cells from DA-3/sec tumors that grew in nude BALB/c animals did not result in tumors in immunologically intact BALB/c mice but did grow when re-inoculated into other nude BALB/c animals (data not shown).



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  		Fig. 2. DA-3/sec tumor formation in immunocompromised mice. A. A total of 1 x 106 DA-3, DA-3/neo, DA-3/TM, or DA-3/sec cells was implanted s.c. into BALB/c nude mice. Tumor formation was monitored using calipers. Data pooled from two separate experiments of four mice per group are shown. By day 7, 100% of mice implanted with DA-3, DA-3/neo, and DA-3/TM were positive for tumors, whereas 100% of mice implanted with DA-3/sec cells were positive between 21 and 28 days. B. Different DA-3 transfectants of MUC1/sec have varying times of appearance in nude mice. Tumor growth in BALB/c nude mice was monitored after s.c. injection of 106 DA-3/neo, DA-3/TM, of three separate transfections of MUC1/sec isoform in DA-3 cells, i.e., DA-3/sec11, DA-3/sec, or DA-3/sec22 cells. Data represents six animals per group.

 
Antitumor Immune Cell Effector Functions.
Natural killer (NK) cells are known to exert cytotoxic activity against tumor targets. We evaluated whether unseparated splenocytes from mice implanted with transfectants of the different MUC1 isoform have innate cytotoxic activity against the NK target cell YAC-1. Fig. 3ACitation shows that splenocytes from BALB/c mice implanted with DA-3/sec cells 3 or 10 days before the assay had a higher level of NK-type cytotoxic activity than those from either control mice or those implanted with DA-3/TM cells.



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  		Fig. 3. Immune function in DA-3/sec-challenged mice. A. Splenocytes derived from DA-3/sec–injected mice have a higher NK activity than normal splenocytes. BALB/c mice were injected with 106 DA-3/sec cells s.c. At days 3 and 10 postinjection, mice were sacrificed along with uninjected age-matched controls, and the spleens were removed. After red blood cell lysis, total splenocytes were incubated with 51Cr-labeled YAC-1 cells for 4 hours at the indicated E:T ratios. B. CD8+ but not CD4+ T cells are cytotoxic effector cells against DA-3/sec tumor cells. Purified CD4+ and CD8+ splenic T cells from animals injected with DA-3/sec cells were double stained with anti-CD4 and anti-CD8 monoclonal antibodies and analyzed by FACScan for purity (left panel). Purified populations of each cell type were tested against DA-3/sec cells at the indicated E:T ratios in a 4-hour 51Cr release assay (right panel). One of three representative experiments is shown.

 
In additional studies, we looked for the presence of specific cytotoxic T cells in mice implanted with either DA-3/TM or DA-3/sec cells. Splenic cells were expanded in vitro in the presence of either DA-3/TM or DA-3/sec cell stimulators and tested against DA-3–transfected cell targets. The data presented in Table 2Citation show that splenocytes from mice that received injections of DA-3/sec cells and stimulated in vitro with DA-3/sec cells were capable of efficiently lysing DA-3/sec target cells. Additionally, splenocytes from animals implanted with DA-3/sec and DA-3/TM cells mixed in a 1:1 ratio and stimulated in vitro with DA-3/sec cells were also capable of lysing DA-3/sec targets. In contrast, the same effector cells with the same in vitro treatment did not lyse DA-3/TM target cells. Furthermore, splenic cells from mice injected with the mixed DA-3/sec:DA-3/TM cells did not lyse either DA-3/TM or DA-3/sec targets after in vitro activation with DA-3/TM tumor cells. These results suggest that antitumor activity is induced by DA-3/sec cells but not DA-3/TM cells.


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  		Table 2 Cytotoxic activity of splenic cells from mice implanted with DA-3/TM, DA-3/sec, or both against tumor targets after in vitro stimulation

 
In other studies, splenic CD4+ and CD8+ T cells were isolated from mice that received injections 2 weeks prior of DA-3/sec cells. These T-cell subsets, which were over 90% pure, were activated in vitro for 5 days with mitomycin C-treated DA-3/sec cells. Fig. 3BCitation shows that CD4+ T cells had no cytotoxic activity against the DA-3/sec cells. In contrast, CD8+ T cells had higher cytotoxic levels against these targets than the unseparated T cells. These results point to CD8+ T cells as the major effectors of DA-3/sec mediated lysis.

Prior Exposure of BALB/c Mice to DA-3/sec Cells Confers Protection against Several Tumor Types.
We evaluated whether previous exposure of BALB/c mice to DA-3/sec cells conferred protection against the MUC1-expressing tumor DA-3/TM or tumor cells containing the selection vector only, DA-3/neo. Before challenge, experimental groups of six animals were inoculated s.c twice with 1 x 106 DA-3/sec cells 2 weeks apart. The challenge consisted of either 1 x 106 DA-3/TM or DA-3/neo cells mixed with 1 x 106 DA-3/sec cells. Six control animals received the tumor challenge alone without previous exposure to DA-3/sec cells.

By 2 weeks after implantation, all of the animals in the control groups had tumors. Importantly, however, the animals that received DA-3/TM tumor cells mixed with DA-3/sec cells after prior exposure to DA-3/sec cells (Fig. 4ACitation , left panel) showed a delay in the time of tumor appearance as compared with the control group, and a substantial number of the animals failed to develop any tumors. Furthermore, this protection did not appear to be due to a recognition of the MUC1 molecule because a similar effect was seen in animals exposed to DA-3/sec cells and challenged with a mixture of DA-3/neo and DA-3/sec mammary tumor cells (Fig. 4ACitation , right panel).



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  		Fig. 4. DA-3/sec cells promote tumor immunity against multiple tumor cell types. A. Mice previously exposed to DA-3/sec cells can reject a challenge of DA-3/TM or DA-3/neo cells mixed with DA-3/sec cells. BALB/c mice received two s.c. injections of 106 DA-3/sec cells 2 weeks apart with no tumors appearing after each inoculation. Two weeks after the third injection, mice were challenged with 106 of either DA-3/TM or DA-3/neo cells mixed with 106 DA-3/sec cells (treated). Controls consist of naïve mice injected with challenge. Percentages of mice developing tumor over time represent the results of two independent experiments of six mice per group. DA-3/TM–challenged mice, controls versus treated (P < 0.001); DA-3/neo–challenged mice, controls versus treated (P = 0.003). B. Protective effect of DA-3/sec cells is not limited to cells of the DA-3 background. A total of 106 K7 osteosarcoma or RENCA cells was injected into BALB/c mice that previously received two injections of DA-3/sec cells. Both naïve (control) and DA-3/sec–injected (treated) mice were challenged with either 106 K7 or RENCA cells mixed with 106 DA-3/sec cells before injection. RENCA-challenged mice, controls versus treated (P = 0.062). K7 osteosarcoma-challenged mice, controls versus treated (P = 0.013). C. Effective antitumor activity can be detected without previous exposure to DA-3/sec cells when a lower number of DA-3/TM cells is mixed with DA-3/sec cells at the time of challenge. Results represent data from two experiments with five animals per group. Mice receiving the mixture of DA-3/TM and DA-3/sec were protected compared with DA-3/TM only controls (P < 0.005).

 
A series of studies were done to evaluate whether pre-exposure to DA-3/sec cells also conferred protection against the growth of tumors other than those of the DA-3 mammary tumor background. The renal cell carcinoma, RENCA, and the K7 osteosarcoma, both syngeneic to BALB/c mice, were used. The BALB/c mice pre-exposed twice, 2 weeks apart, to the DA-3/sec cells, followed by a challenge with a mixture of RENCA cells and DA-3/sec cells showed a 40% reduction in the number of animals developing tumors as compared with the untreated control mice receiving the same inoculation (Fig. 4BCitation , left panel). As was the case with DA-3/TM and DA-3/neo cells, the growth of RENCA cells was not impaired unless they were mixed with the DA-3/sec cells at the time of challenge, even if the animals had been previously exposed to DA-3/sec cells. Similar results were obtained in experiments with the K-7 cells (Fig. 4BCitation , right panel), where 60% of the animals failed to develop tumors.

In another experiment, animals that were pre-exposed twice to DA-3/sec cells and then challenged with 1 x 106 DA-3/TM cells but not DA-3/sec cells showed only a short time delay in time of tumor appearance, i.e.,13 of 22 animals had palpable tumors at 2 weeks, whereas, in the control group of untreated animals, inoculated with 1 x 106 DA-3/TM cells, 13 of 14 had tumors at 1 week. However, we found that implantation of a lower number of DA-3/TM cells (5 x 105) mixed with 106 DA-3/sec cells appeared to afford protection against growth of the tumor even when the mice had not been pre-exposed to DA-3/sec cells (Fig. 4C)Citation . These results indicate that mixing of challenge with DA-3/sec is important for overall protection against tumor formation and that pre-exposure of mice to DA-3/sec does not appear to be necessary for protection if the lower numbers of tumor cells are used in the challenge.

A Novel 11 Amino Acid Peptide Is Present at the COOH Terminus of MUC1/sec.
A possible clue as to why the secreted form of MUC1 confers antitumor protection was obtained from analysis of the sequences of the MUC1 transmembrane and secreted isoforms. Fig. 5ACitation is a schematic drawing of the MUC1/TM and MUC1/sec isoforms. The sequences are depicted from the 5'-termini at the left of the figure and the different areas are identified as follows: tandem repeat region (TR), the signal peptide coding region (SP), and the transmembrane and cytoplasmic domains are indicated by TM and CYT, respectively. The secreted form lacks the TM and CYT regions and instead is terminated by a unique sequence coding for an 11 amino acid peptide (VSIGLSFPMLP; ref. 23 ). A high-stringency BLASTP (24) analysis of these 11 amino acids revealed no identity with any other known protein sequence. At low stringency, there are some similarities with glutathione S-transferase from two Echinococcus species: to the hypothetical protein MLCB4.30 of Mycobacterium leprae and to EF hand protein of two plant species.



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  		Fig. 5. The MUC1/sec-derived peptide, immunoenhancing peptide (IEP), shows antitumor properties. A, schematic representation of human transmembrane MUC1 (MUC1/TM) and human-secreted MUC1 (MUC1/sec) isoforms. Note that the MUC1/sec isoform lacks the transmembrane and cytoplasmic regions but still encodes the tandem repeats. A unique segment encoding for an 11 amino acid peptide (IEP) not found in MUC1/TM is present at the 3'-terminus of the MUC1/sec cDNA. B. Pre-exposure to IEP can protect mice from DA-3/TM and DA-3/neo tumor growth. Mice received two injections of KLH-IEP as described in Materials and Methods before challenge with DA-3/TM or DA-3/neo cells mixed with KLH-IEP before injection (treated). Control mice represent naïve mice challenged with tumor cells + KLH-IEP. DA-3/TM–challenged mice, controls versus treated (P = 0.1). DA-3/neo–challenged mice, controls versus treated (P = 0.014). C, protective effect of MUC1/sec-derived peptide against tumors originating from different backgrounds and strains. Tumor cells were injected into mice that received two injections of KLH-IEP as described in Materials and Methods. RENCA-challenged mice, controls versus treated (P = 0.113). Lewis lung carcinoma (LLC) challenge, controls versus treated (P < 0.001). D. IEP synthesized as a multiple antigenic peptide-IEP is effective at protecting BALB/c mice from tumor formation. BALB/c mice received three injections of either multiple antigenic peptide-IEP or with a scrambled version of IEP synthesized as a multiple antigenic peptide (multiple antigenic peptide-scrambled), followed by challenge with 106 DA-3/TM tumor cells (P = 0.003) or DA-3/neo tumor cells (P = 0.014) mixed with the appropriate multiple antigenic peptide conjugate. Each experiment represents five mice per group.

 
A chicken IgY antibody specific for the unique 11 amino acid portion of MUC1 was prepared to confirm the presence of this peptide in the DA-3 cells transfected with the secreted MUC1 isoform. A direct ELISA was also developed using this reagent to detect the secreted peptide in the supernatants of DA-3/sec cultures. All DA-3/sec cultures tested were positive for this molecule, whereas no expression of the peptide was found in the DA-3, DA-3/neo, or DA-3/TM cell supernatants (data not shown).

The MUC1/sec-derived Peptide Protects BALB/c and C57BL/6 Mice against Several Tumor Types.
Experiments were done to evaluate the effectiveness of the peptide instead of DA-3/sec cells as a protective agent. The initial experiments used synthesized peptide conjugated to KLH. Experimental mice received two injections 8 days apart of the peptide- KLH in incomplete Freund’s adjuvant. Eight days after the second injection, the animals were challenged with tumor cells mixed with the peptide-KLH. Only 60% of the animals challenged with either DA-3/TM cells or DA-3/neo cells mixed with the peptide-KLH developed tumors (Fig. 5B)Citation . In both experiments, 100% of the control mice receiving no prior exposure to peptide-KLH developed tumors within 2 weeks (Fig. 5B)Citation . Importantly, in the peptide-KLH–protected animals, the survivors showed no signs of tumor 6 months after challenge with either DA-3/TM or DA-3/neo cells. KLH alone had no effect on tumor growth because 100% of the mice that received a challenge of 106 DA-3/TM or DA-3/neo cells formed tumors in the presence or absence of KLH.

These results were not due to a toxic effect of the peptide, which we have named immunoenhancing peptide, as direct addition of this molecule to cultured DA-3, DA-3/TM, DA-3/sec, or DA-3/neo tumor cells did not affect their in vitro growth (Table 3)Citation . Furthermore, culturing the cells with peptide for 4 days before implantation did not affect their potential to produce tumors in BALB/c mice. Tumor cells treated in this manner grew with the same in vivo kinetics as cells that had never been exposed to this molecule. In additional studies, we evaluated the in vivo protective effect of peptide pretreatment against two other tumors. Fig. 5CCitation , left panel, shows that pre-exposure to immunoenhancing peptide-KLH afforded only minimal protection against challenge of RENCA cells mixed with peptide-KLH in BALB/c mice. However, as seen in Fig. 5CCitation , right panel, when the immunoenhancing peptide-KLH was used twice as pretreatment in another mouse strain, C57BL/6, and the animals were subsequently challenged with Lewis lung carcinoma cells mixed with peptide, 50% of the animals were protected against tumor development. These results suggest that the effect of MUC1/sec-derived peptide is not limited to one particular strain of mouse.


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  		Table 3 Effect of the MUC1/sec unique peptide (IEP) on in vitro tumor growth

 
In addition to the peptide-KLH conjugation, we synthesized immunoenhancing peptide as a multiple antigenic peptide (multiple antigenic peptide). Either immunoenhancing peptide or a scrambled sequence was conjugated to a branched lysine core with four peptides per molecule. The multiple antigenic peptide configuration has several advantages over a KLH conjugation, including higher immunogenicity, smaller volumes of peptide to inject, and, importantly, we have found that no adjuvant is required.

BALB/c mice were pre-exposed to either the multiple antigenic peptide-immunoenhancing peptide or multiple antigenic peptide-scrambled as described in the Materials and Methods. Subsequently the mice pre-exposed to the corresponding peptide were challenged with DA-3/TM or DA-3/neo (Fig. 5D)Citation tumor cells mixed with either multiple antigenic peptide-immunoenhancing peptide or multiple antigenic peptide-scrambled. Interestingly, the multiple antigenic peptide-immunoenhancing peptide successfully protected ~50% of mice challenged with either DA-3/TM or DA-3/neo. In contrast, the multiple antigenic peptide-scrambled form failed to elicit any protection against tumor growth. These results suggest that immunoenhancing peptide can be used to delay and/or prevent tumor cell growth in vivo.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study identifies an 11 amino acid peptide derived from the COOH terminus of MUC1/sec as an immunostimulatory molecule capable of preventing tumor formation in murine models of cancer. The failure of DA-3 mammary adenocarcinoma cells transfected with MUC1/sec to grow in vivo led us to additionally explore this protein. MUC1/sec, an alternative splice variant of MUC1, is secreted because it lacks both the transmembrane and cytoplasmic tail of MUC1, and the 3'-end of the MUC1/sec transcript encodes for a unique 11 amino acid peptide. The fact that DA-3 cells transfected with full-length MUC1 grow with similar kinetics as parental tumors in vivo suggests that unique properties of MUC1/sec contribute to its inability to establish tumors in vivo.

Both humoral and adaptive immune responses against MUC1 have been detected in various model tumor systems, as well as in cancer patients (25) . MUC1, which is expressed by a variety of epithelial tissues, is highly glycosylated on normal cells masking the antigenic epitopes of the core protein. In contrast, in cancer cells of various origins, MUC1 is overexpressed and hypoglycosylated. This allows the immune system to recognize the normally masked antigenic mucin sites on tumor cells without major targeting of normal cells.

Earlier studies by Finn et al. (26) with lymphocytes from pancreatic patients showed that MUC1 is recognized by both CD4+ and CD8+ human T cells as a cell surface molecule directly through the T-cell receptor but independent of MHC class I or class II molecules. Similar results were later observed by these investigators in other tumor types (27) . Thus, cytotoxic T cells from malignant ovarian tumors were found to recognize polymorphic epithelial MUC1 core peptides (28) . Interestingly, cytotoxic T cells in mice immunized with human MUC1 are MHC restricted (27) .

In our system, NK and CD8 T cells may be playing an in vivo role in DA-3/sec tumor cell lysis. Unpurified splenocytes from BALB/c mice implanted with DA-3/sec tumor cells have higher NK activity against YAC-1 targets than normal BALB/c mice, and this increased cytolytic activity is enhanced upon longer in vivo exposure to DA-3/sec cells. NK cells are classic antitumor effector cells and can be activated through the ligation of multiple surface receptors and through cytokine stimulation (29) . Exposure to DA-3/sec cells may induce activation of NK cells through direct cell-cell interactions, or, alternatively, indirect activation may occur by stimulating cytokine secretion by antigen-presenting cells. The ability of DA-3/sec-primed CD8 T cells to lyse DA-3/sec tumor cells in vitro was also showed (Table 2)Citation . In contrast, DA-3/TM-primed effector cells could not lyse DA-3/TM targets, suggesting that the DA-3/sec tumor cells may be more immunogenic or have an increased susceptibility to lysis than parental or MUC1-transfected DA-3 cells. T-Cell immunity is thought to be a major driving force in the rejection of tumor cells (25) . Indeed, many tumor vaccination models, including those aimed at using the MUC1 antigen, attempt to specifically invoke Th1 immunity in the hopes of generating tumor-specific T cells (12 , 30 , 31) . It is clear that DA-3/sec cells elicit T-cell immunity; however, what role the MUC1/sec-derived peptide plays in the generation of Th1 immunity is still unclear.

Several immunostimulatory molecules and adjuvants can inherently induce Th1 immunity. For example, bacterial oligodeoxynucleotides containing specific motifs centered on a CpG dinucleotide (CpG-ODN) have been routinely used to induce CTL effectors (32 , 33) . Several mechanisms are induced upon CpG-ODN stimulation, including local cytokine and chemokine expression that result in activation of components of the innate immune system such as NK cells. CpG-ODN stimulates a nonspecific antitumor response by binding Toll-like receptor 9, resulting in the activation of antigen-presenting cells through enhanced processing of antigen and secretion of cytokines (34 , 35) . A recent study, however, has revealed repeated injections of CpG in mice result in multifocal liver necrosis and immunosuppression (36) . Cytokines, such as granulocyte macrophage colony-stimulating factor, have also been used as immunostimulatory agents to stimulate antitumor immunity (37) . As with CpG-ODN and many cytokines, the MUC1/sec-derived peptide, immunoenhancing peptide, clearly enhances antitumor activity. Although the precise mechanism of immunoenhancing peptide is currently unclear, preliminary data suggest that neither immunoenhancing peptide nor DA-3/sec tumor cells do not stimulate the functional maturation of bone marrow-derived dendritic cells in vitro, nor do they stimulate proinflammatory cytokine stimulation from dendritic cells,3 in contrast to CpG-ODN and cytokine adjuvants. However, mice inoculated with DA-3/sec cells have increased NK activity, and currently, we are investigating whether immunoenhancing peptide alone can also elicit this response. Although many adjuvants can have substantially negative side effects, no adverse effects, including weight loss or autoimmune responses, were observed in immunoenhancing peptide-treated animals.

Interestingly, the immunostimulatory activity is encoded within a mucin protein. Although this is a unique property, another mucin, MUC7, has been shown to encode a 20 amino acid peptide that has potent antimicrobial properties (38 , 39) . Because the mucin proteins are found at many interfaces between the host cells and external environments, it is logical to conclude that this protein family, while serving as physical barriers, may also encode properties capable of stimulating an immune response.

Here, we show that mice previously exposed to MUC1/sec-expressing cells or a MUC1/sec-derived peptide are able to slow or, in some cases, prevent subsequent growth of several tumor types. One caveat of these experiments is the fact that, to date, the challenge must be co-injected with either DA-3/sec cells or the MUC1/sec-derived peptide for the antitumor effect to be substantial. Work on alternative vaccine protocols and therapeutic applications are ongoing in an attempt to solve this problem.

The total protection or tumor retardation observed in mice previously exposed to DA-3/sec cells and then challenged with DA-3/TM cells could be attributed to previous exposure to the entire MUC-1 molecule. However, animals challenged with DA-3/neo cells were equally protected, indicating that the effect was not that of a classic antigen recognition vaccination protocol. This conclusion was further strengthened when similar effects were observed in mice pre-exposed to DA-3/sec cells and challenged with the RENCA renal carcinoma and the K7 osteosarcoma. Although these tumors are syngeneic to BALB/c mice, they are unrelated to the DA-3 mammary tumor background. One could hypothesize that the unique peptide of MUC1/sec may be functioning, in part, as an immunoadjuvant because, at the time of challenge, the tumor cells had to be mixed with DA-3/sec cells to observe the antitumor effect. Indeed, the hydrophilic nature of the peptide suggests that it may be highly immunogenic. Thus, the presence of these 11 amino acids in the cells transfected with MUC1/sec may be crucial for the antitumor effects observed.

The immunologic mechanisms involved in the absolute protection against tumor growth observed when the DA-3/sec cells are initially inoculated in untreated BALB/c mice may not be identical to those observed in the vaccination-like protocols with the peptide. There appears to be no antigenic specificity afforded by the MUC-1/sec in the latter, although the MUC1 molecule has been used in various classic antigen-specific studies (40, 41, 42, 43) . Indeed, there may be additional contributions from other domains of the secreted mucin molecule, such as the tandem repeats, conferring protection against the initial growth of DA-3/sec cells in immunocompetent mice. However, the in vivo protocols, with the peptide in the absence of other portions of the secreted MUC1, results in retarded tumor appearance or absence of growth, strongly suggesting that the unique peptide can per se play a significant role in the antitumor process. Ongoing experiments include the generation of DA-3 cells that express MUC1/sec lacking the tandem repeat portion of the gene to additionally assess the role of the MUC1/sec protein in tumor development.

Determinant spreading is the process in which antigens that are distinct and non cross-reactive with the stimulating antigen become additional targets of the induced immune response. The process of determinant spreading has been observed during clearance of infectious diseases, in response to allotranplants, and during human immunotherapy trials (44) . The MUC1/sec-derived peptide (immunoenhancing peptide) is capable of stimulating an immune response against multiple tumor targets across MHC backgrounds, highlighting the possibility that immunoenhancing peptide may be an immunodominant epitope resulting in the lysis of tumor cells that are presenting immunoenhancing peptide. The resulting lysis would then lead to an environment in which antigen presenting cells are stimulated to present tumor-specific antigens that ultimately lead to the prevention of tumor cell growth.

As mentioned previously, the tumor challenge needed to be mixed with immunoenhancing peptide or DA-3/sec cells to have a significant decrease in growth. This may suggest that the mechanism of action of the MUC1/sec protein or immunoenhancing peptide occurs within the tumor cell itself and not on the host immune system. Alternatively, the MUC1/sec or immunoenhancing peptide may act on the surrounding tissue, including epithelial cells, inducing secretion of proinflammatory cytokines and other factors that would promote antitumor responses. We are currently characterizing the cell types and mechanisms involved in the immunoenhancing potential of immunoenhancing peptide.

The results of the present study suggest, however, that this peptide may be helpful alone or in combination with other molecules against not only a wide variety of tumors but also in cases when immunostimulation against various pathogens is desirable.


   FOOTNOTES
 
Grant support: NIH Grant R01 CA25583 and Viragen.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Diana M. Lopez, Department of Microbiology and Immunology, University of Miami-School of Medicine, P. O. Box 016960 (R-138), Miami, FL 33101. Phone: (305) 243-6632; Fax: (305) 243-4409; E-mail: dlopez{at}med.miami.edu

3 J. Grosso, unpublished observations. Back

Received 3/ 9/04. Revised 8/18/04. Accepted 8/26/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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