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Cancer Research 67, 1887-1892, March 1, 2007. doi: 10.1158/0008-5472.CAN-06-3960
© 2007 American Association for Cancer Research
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Priority Reports

Immunoediting Sculpts Tumor Epitopes during Immunotherapy

Reshma Singh and Yvonne Paterson

Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania

Requests for reprints: Yvonne Paterson, Department of Microbiology, University of Pennsylvania School of Medicine, 323 Johnson Pavilion, 36th Street and Hamilton Walk, Philadelphia, PA 19104-6076. Phone: 215-898-3461; Fax: 215-573-4666; E-mail: yvonne{at}mail.med.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunoediting of tumor-associated antigens occurs in response to immune pressure. We show that the mutation of residues within epitopes of HER-2/neu leads to the outgrowth of autochthonous tumors after immunizing HER-2/neu transgenic mice with Listeria monocytogenes therapeutic vaccines expressing fragments of HER-2/neu. Three of these vaccines target the extracellular domain (LmLLO-EC1, LmLLO-EC2, and LmLLO-EC3), and two of these vaccines target the intracellular domain (Lm-LLO-IC1 and Lm-LLO-IC2). Mutations occurred in the regions of the HER-2/neu molecule targeted by the Listeria strain expressing that region, which suggests that the rate of generation of escape mutants was a significant factor in the efficacy of each vaccine. A longer delay in the onset of tumors after immunotherapy occurred with the vaccine that targeted the kinase domain. We verified that the mutations in this domain occurred within novel CD8+ T-cell epitopes, and that the mutation of these residues abrogated CTL responses to these epitopes. The long delay in the onset of tumors after immunotherapy targeting the kinase domain may be because this region of HER-2/neu cannot undergo extensive mutations without impairing its ability to signal cell growth. [Cancer Res 2007;67(5):1887–92]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cancer immunotherapies are often effective for a limited time due to escape mechanisms that include down-modulation of the tumor antigen (1), decrease in MHC class I expression (2), induction of clonal anergy of tumor-specific T cells (3), and alterations in the antigen presentation pathway (4). Escape mechanisms involving changes in expression of the tumor antigen may be the result of immunoediting, the alteration of tumor immunogenicity due to the antitumor response of the immune system (5). The study of these changes with the tumor antigen HER-2/neu has previously focused on the down-regulation of the tumor antigen, and neu-negative variants have been described in response to tumor-specific vaccination (1).

HER-2/neu, a 185-kDa glycoprotein and a member of the epidermal growth factor (EGF) family of receptors, is overexpressed in 20% to 40% of all breast cancers (6). Normal epithelial cells express the HER-2/neu gene in a single copy, which is amplified in malignant cells. Overexpression may contribute to the initiation and progression of disease (7). In a mouse model of breast cancer, where the rat HER-2/neu gene is under the control of the mouse mammary tumor virus promoter (8), female mice develop autochthonous mammary tumors between 4 and 9 months of age. Growth of tumors is due to the overexpression and enhanced signaling of HER-2/neu.

We have described Listeria monocytogenes–based vaccines expressing overlapping fragments of HER-2/neu that are equally capable of inducing stasis in the growth of transplanted tumors in wild-type FVB/N mice (9) and HER-2/neu transgenic mice (10). We now show that these vaccines differ in delaying the onset of autochthonous tumor growth in transgenic mice. The eventual growth of the tumors seems to be related to specific mutations in epitopes targeted by the Lm-LLO-HER-2/neu vaccines, which destroy their ability to be recognized by CTLs.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. FVB/N HER-2/neu transgenic mice were housed and bred at the Veterans' Administration Hospital at the University of Pennsylvania. Experiments on mice were done in accordance with regulations by the Institutional Animal Care and Use Committee of the University of Pennsylvania and the Veterans' Administration.

Listeria vaccine strains. Strains used are Lm-LLO-EC1, Lm-LLO-EC2, Lm-LLO-EC3, Lm-LLO-IC1, and Lm-LLO-IC2 (9). A L. monocytogenes–based vaccine for NY-ESO-1 was provided by Dr. Paulo Maciag and used as a control for antigen specificity. Bacteria were grown in BHI medium (BD, Sparks, MD) with 50 µg/mL chloramphenicol and frozen in 1 mL aliquots at –80°C. For injection, the vaccines were washed twice with sterile PBS and resuspended in PBS.

Peptides. Peptides were synthesized by EZBiolab, Inc. (Westfield, IN). The sequences are GSGAFGTVYK (728–737), AFGTVYKGI (731–739), TSPKANKEI (760–768), PYVSRLLGI (781–789), STVQLVTQL (793–801), KITDFGLARL (861–870), and RPRFRELVSE (967–976). The sequences for the mutated epitopes are GSGAFGTVAK, AFGTVAKGI, DSPKANKEI, PAVSRLLGI, STVQVTQL, KITDFGLARR, and RPRFRELLSE.

Autochthonous tumor protection. Twelve to 15 mice per group were vaccinated with 0.1 LD50 (9) of each vaccine or Lm-LLO-NYESO-1 or PBS as controls. Statistical analysis of differences in autochthonous tumor growth was done using the Kaplan-Meier log-rank test using SPSS 11 (SPSS, Inc., Chicago, IL), comparing the time of onset of tumor growth between each vaccine group and the control groups.

Analysis and mapping of mutations. Tumors were excised, and DNA was extracted from the samples using a Puregene Genomic DNA Purification kit (Gentra Systems, Minneapolis, MN). Sequencing was done by the University of Pennsylvania Sequencing Facility and then analyzed using Sequencher 3.0 (Gene Codes Corp., Ann Arbor, MI). Mutations that did not occur in three PCR reactions were discarded as PCR-induced mutations. Molecular modeling was done using PyMol (DeLano Scientific, LLC, San Francisco, CA).

Cell lines. The FVB/N syngeneic NT-2 tumor cell line has been described previously (11). The 3T3 cell line is a mouse fibroblast line that expresses MHC class I molecules of the "q" haplotype. The propagation of NT-2 and 3T3 cells has been previously described in detail (10–12).

CTL assays. Six-week-old female HER-2/neu transgenic mice were vaccinated with 0.1 LD50 of Lm-LLO-IC1 or 200 µL PBS. Splenocytes were used in a standard CTL assay with 3T3 target cells loaded with 1 µg/mL of the target peptide as described previously (10, 12).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Delay in tumor growth following protein tyrosine kinase vaccination. The HER-2/neu protein tyrosine kinase domain is contained within the Lm-LLO-IC1 vaccine (9). This region of the rat neu gene is >98% homologous to both mouse and human neu genes, which should result in immune tolerance to this region (13). Thus, we did not expect the Lm-LLO-IC1 vaccine to be effective in preventing the development of autochthonous tumors in FVB/N HER-2/neu transgenic mice. The Lm-LLO-IC1 vaccine significantly (P = 0.00001) delayed the onset of tumor growth compared with control groups. The first mouse in this group developed a tumor in week 36, at least 10 weeks after tumor onset began in all of the other groups (Fig. 1 ). At this point, the Lm-LLO-IC1 vaccine group was significantly better at delaying tumor growth than the other HER-2/neu vaccines (P = 0.0086 for Lm-LLO-IC1 compared with Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-IC2 and P = 0.0003 compared with Lm-LLO-EC3). The vaccines segregated in three groups, with Lm-LLO-IC1 as the most efficacious, Lm-LLO-EC3 (P = 0.5685) as the least, and a middle group of Lm-LLO-EC1 (P = 0.0036), Lm-LLO-EC2 (P = 0.0062), and Lm-LLO-IC2 (P = 0.0054), which were equally effective. The antitumor efficacy of these vaccines was not permanent, as tumors grew in all mice in each group by 1 year. With the transplantable tumors, effects on tumor growth were observed over a relatively short period (~7 weeks; refs. 9, 10) compared with this study where the emergence of autochthonous tumors occurs after 6 months.


Figure 1
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  		Figure 1. Lm-LLO-HER-2/neu vaccines delay the onset of autochthonous tumor growth in FVB/N HER-2/neu transgenic mice. FVB/N HER-2/neu transgenic mice were vaccinated before the onset of palpable tumor growth beginning at 6 wks of age. Mice were again vaccinated at 9, 12, 15, and 18 wks of age and checked by palpation every 2 d for the onset of tumor growth. Groups were examined in a single experiment. They have been separated for readability. A, PBS and Lm-LLO-NYESO1. B, Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-EC3. C, Lm-LLO-IC1 and Lm-LLO-IC2.

 
Specific mutations in HER-2/neu following Lm-LLO-HER-2/neu vaccination. The delayed development and subsequent outgrowth of tumors led us to examine tumor escape from the anti-HER-2/neu immune responses generated after vaccination. More than 60% of the cells in the tumors that grew out in our study retained surface expression of HER-2/neu as determined by fluorescence-activated cell sorting analysis (data not shown); thus, we analyzed the HER-2/neu gene to determine if the oncogene was mutating to escape immune detection. In addition to the wild-type sequence, HER-2/neu from autochthonous tumors that grew in the transgenic mice following vaccination with the Lm-LLO-HER-2/neu vaccines contained specific mutations in the region corresponding to the vaccine (Table 1 ). The majority of the tumor cells in each tumor contained mutations as there was a distinct peak for the mutated residue, but not every tumor cell contained each mutation as there was an underlying, smaller peak for the wild-type residue. There was a low level of random mutation in each tumor in the other regions and, on average, a 10-fold increase in the frequency of mutations occurring in the vaccine target region versus the appearance of a random mutation (Supplementary Table S1). Thus, tumors seem to be mutating to escape immune pressure. A number of CTL epitopes have been identified in HER-2/neu for the FVB mouse (10–12), and each of these epitopes contained at least one mutation (Supplementary Fig. S1).


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  		Table 1. Specific mutations occur in autochthonous tumors that develop after the vaccination of FVB/N HER-2/neu transgenic mice

 
The tyrosine kinase domain of HER-2/neu is highly homologous to other kinase domains; thus, it was not expected to be targeted by the immune system due to tolerance mechanisms. Surprisingly, the kinase domain of HER-2/neu (716–980) mutated in response to vaccination but did not accumulate random mutations in the absence of Lm-LLO-IC1 vaccination (Table 1). Although Lm-LLO-IC1 expresses the largest fragment of HER-2/neu (392 residues) compared with the other five vaccines (307 for Lm-LLO-EC1, 199 for Lm-LLO-EC2, 177 for Lm-LLO-EC3, and 240 in Lm-LLO-IC2), it did not accumulate significantly more mutations. Only a few of these (Y736A, T760D, Y782A, L797 deleted, L870R, and V974L) appeared in the tyrosine kinase domain and are marked in bold (Table 1). Specific mutations support the hypothesis that tumors mutate to escape immune pressure and may be limited in their ability to mutate in regions essential for the function of HER-2/neu, such as the kinase domain.

Kinase domain mutations are all within CD8+ T-cell epitopes, and mutations abrogate CTL responses. We sought to determine if the mutated residues identified from the mutational mapping were mutating because they were CTL targets. We examined the sequence of IC1 for putative epitopes containing these mutations and generated peptides using the Rankpep predictor1 for the "d" haplotype. H-2d was used as a predictor because of its structural similarities to H-2q (14). When these peptides were pulsed onto target cells in a CTL assay, the targets were recognized and killed by CD8+ T cells from vaccinated mice (Fig. 2 ). These novel epitopes are GSGAFGTVYK (728–737), AFGTVYKGI (731–739), PYVSRLLGI (781–789), TSPKANKEI (760–768), RPRFRELVSE (967–976), STVQLVTQL (793–801), and KITDFGLARL (861–870). Each of the six mutations tested alters a residue that is contained within one or more of the novel CD8+ T-cell epitopes. To test if mutations in the CTL epitopes allowed for immune escape from CTLs generated by Lm-LLO-IC1 vaccination, we compared the ability of CTLs from immunized mice to kill target cells pulsed with peptides representing the epitopes with the native HER-2/neu sequence with those with the mutated sequence. Mutation of the individual residues within these epitopes abrogated the ability of CTLs to kill peptide pulsed target cells (Fig. 2).


Figure 2
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  		Figure 2. Each of the residues mutated in the kinase domain corresponds to a residue within a CD8+ T-cell epitope. Splenocytes were used for an in vitro CTL assay. Each ratio was set up in triplicate. Total lysis of chromium-loaded target cells was stimulated by the addition of 2% Triton X-100. Percent specific lysis was calculated: % = 100 [(experimental lysis – spontaneous lysis) / (total lysis – spontaneous lysis)]. {diamondsuit}, splenocytes from PBS-vaccinated mice; {blacksquare}, splenocytes from Lm-LLO-IC1–vaccinated mice, each with target cells presenting non-mutated peptides; {blacktriangleup}, splenocytes from PBS-treated mice; bullet, splenocytes from Lm-LLO-IC1–vaccinated mice, each with target cells presenting mutated peptides. The wild-type peptides that were pulsed onto the target cells are shown with the graphs, and the mutated residue is highlighted in bold. The residue that was mutated and the residue to which it was mutated are shown below the wild-type sequence. The peptides used are as follows: GSGAFGTVYK and GSGAFGTVAK, AFGTVYKGI and AFGTVAKGI, PYVSRLLGI and PAVSRLLGI, TSPKANKEI and DSPKANKEI, RPRFRELVSE and PRPFRELLSE, STVQLVTQL and STVQVTQL, and KITDFGLARL and KITDFGLARR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tolerance is an important hurdle to overcome with cancer immunotherapy. As HER-2/neu is a self-antigen in the HER-2/neu transgenic mouse, it may have deleted or anergized high-affinity HER-2/neu reactive T cells (15). Furthermore, antitumor immune responses are dampened by CD4+CD25+ regulatory T cells (10). It has been shown that the intracellular domain of HER-2/neu containing the kinase domain can be safely used as a vaccine (16). Our studies support the idea that the kinase domain can be an effective target in a cancer vaccine, and that the intracellular domain of HER-2/neu can be highly immunogenic.

During viral infections, both antibodies and CTLs can select for escape mutants through the alteration of one or more amino acids, a mechanism called antigenic drift (17). A similar process for tumor growth is described as immunoediting and is the result of the immune system selecting for tumors that it can no longer target (18). Immunoediting in vivo has been seen in the P1A tumor model where recurrent tumors have mutated the 35-43 P1A CTL epitope after passive immunotherapy with high doses of P1A TCR transgenic T cells (19). Our study is the first to show that CTL escape mutants can arise in tumors during the hosts' own immune response. Treatment of mice with Listeria-based vaccines for HER-2/neu leads to the growth of tumors that have altered amino acid sequences in regions that destroy CD8+ T-cell epitopes. This tumor immunoediting generates tumors that escape immune detection. This is the first time such a phenomena has been seen in the FVB/N HER-2/neu transgenic mouse model, or on such a large scale with such a wide variety of mutations, 65 in total, in vivo in any model.

Although the intracellular domain of HER-2/neu has not been crystallized, the similarity in sequences of the tyrosine kinase domain of HER-2/neu with all other tyrosine kinase domains allows the use of the EGF receptor (EGFR) kinase domain as a model for that of HER-2/neu (20). Figure 3 shows a model with the position of six of the mutations that we observed in the kinase domain of rat HER-2/neu modeled on a human EGFR tyrosine kinase domain (Fig. 3). Interestingly, there were no mutations within the catalytic subunit possibly because such mutations could alter the signaling capabilities of HER-2/neu and result in an impairment of tumor growth. The majority of the mutations also lay outside the ATP-binding domain, in regions that are probably less essential to the function of the kinase domain. The mutations that are the most likely to alter the function of the kinase domain are mutations 1 and 4, which are in ß-sheets within the ATP-binding domain. Because these tumors grow out, these mutations do not seem to abrogate the function of the kinase domain but may alter the binding of ATP. ATP may still bind and be catalyzed to allow for signaling, but the efficiency of these processes could be reduced and decrease the signaling ability of HER-2/neu. Tumors targeted by Lm-LLO-IC1 would grow out after a delay due to decreased signaling and a subsequent decrease in the proliferative capacity of the tumor cells.


Figure 3
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  		Figure 3. Mutations mapped onto the structure of a model of the rat HER-2/neu protein tyrosine kinase domain. Fourteen tumors, two from each vaccine group and control group, were removed when the tumor had reached a size of 2 cm after tumor onset, for DNA extraction. The entire gene was PCR amplified in triplicate and sequenced. Mutations that did not occur in three PCR reactions were discarded as PCR-induced mutations. The mutations were then mapped onto a model of the rat HER-2/neu protein tyrosine kinase domain. The ATP-binding and catalytic domains are identified. *, binding positions of ATP. Mutations identified are (1) Y736A, (2) T 760D, (3) Y782A, (4) L797 deleted, (5) L870R, and (6) V974L. This model suggests an explanation as to why the Lm-LLO-IC1 vaccine is so effective in delaying the onset of autochthonous tumor growth as opposed to the other Lm-LLO-HER-2/neu vaccines.

 
Our findings have implications for improving cancer immunotherapy and preclinical testing strategies. For an autochthonously arising tumor in an animal where the target antigen is a self-protein, similar to human cancers, there is a clear difference in vaccine efficacy between the Lm-LLO-HER-2/neu vaccines. In a transplantable tumor model, all five vaccines are equally efficacious (9, 10), suggesting that transplantable tumor models, which are frequently used for preclinical testing, may not be a reliable indicator of vaccine efficacy. In addition, our study suggests that a cancer immunotherapy approach that shifts immune pressure to epitopes in a different region of the molecule could provide a more effective therapeutic outcome. This study shows that cancer immunotherapies can force the editing of oncogenes, even in domains necessary for signaling. Beyond basic immunosurveillance, this is the first proof that therapeutic vaccines can cause these specific changes in tumors.


   Acknowledgments
 
Grant support: Department of Defense Breast Cancer Research Program Predoctoral Fellowship W81XWH-04-1-0338 (R. Singh) and Cancer Research Institute training grant "Predoctoral emphasis pathway in tumor immunology."

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.

We thank Dr. Ramachandran Murali (University of Pennsylvania) for his assistance mapping the mutations onto the three-dimensional structure of HER-2/neu and Dr. Carl June (University of Pennsylvania) for critically reading the article.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1 http://bio.dfci.harvard.edu/Tools/rankpep.html Back

Received 10/27/06. Revised 12/20/06. Accepted 1/ 3/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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