Introduction
MVA-BN
®-HER2 is currently being developed as a candidate for breast cancer immunotherapy and has shown biological activity in ongoing Phase I clinical trials (NCT0048277). MVA-BN
®-HER2 is a Modified Vaccinia Ankara-based recombinant vaccine vector derived from MVA-BN
® (IMVAMUNE
®), a highly attenuated clonal virus currently in development as a smallpox vaccine [
1]. MVA-BN
®-HER2 encodes a modified form of human epidermal growth factor receptor 2 (HER-2), referred to as HER2. HER2 comprises of the extracellular domains of HER-2 and was further modified to encode two promiscuous T helper cell epitopes from tetanus toxin. This modification has been shown to improve its immunogenicity in a tolerant environment [
2].
Successful cancer immunotherapy will most likely require the induction of comprehensive innate and Th1 adaptive immune responses that provide effector mechanisms able to directly kill tumor cells while combating the inflammatory/immunosuppressive responses induced by the growing tumor. In this regard, MVA-BN
® has been shown to confer protection in animal models of smallpox through the induction of strong Th1 adaptive as well as innate immune responses [
3‐
7]. These intrinsic immune properties are beneficial features to mediate adequate immune responses to MVA-BN
®-encoded transgenes expressing tumor targets. This was demonstrated herein, by comparing the induction of HER-2-specific immune responses mediated by either MVA-BN
®-HER2 or HER2 formulated in Freund’s adjuvant. When tested in a murine model of experimental pulmonary metastasis (CT26-HER-2), MVA-BN
®-HER2-mediated immune responses translated into potent anti-tumor efficacy, while HER2 formulated in Freund’s adjuvant had only modest effects.
Multiple cell types can exert immunosuppressive functions within tumors, thereby hampering the development of successful immunotherapies [
8‐
12]. Among these, regulatory T cells (T
reg) appear to play a critical role in the progression of a number of cancers including breast cancer [
13‐
17]. Even if tumor-specific effector cells are initially recruited into the tumor, the recruitment of T
reg cells into the same site can result in an unproductive anti-tumor response, and this is believed to play a major part in the failure of many cancer immunotherapies presently in development [
14,
18]. Furthermore, recent research in mouse and human showed that, regardless of the overall number of T
reg cells in the tumor, the local balance of T
reg to effector T cells (T
eff) is critical to the success of immunotherapy and ultimately survival [
19‐
22]. The murine CT26 colon carcinoma cell line has been shown to be a potent inducer of immunosuppressive mechanisms [
23‐
25] making it a good model to evaluate the interplay between anti-tumor immunity and tumor-induced immunosuppression. When injected intravenously (i.v.), CT26 cells give rise to experimental pulmonary metastasis while mediating the infiltration of tumor-bearing lungs by a large number of IL-10-secreting T
reg [
25]. Data described here demonstrate that MVA-BN
®-HER2 treatment induced Th1-dominated HER-2-specific cellular and humoral immunity, reduced the amount of T
reg cells in the tumor and altered the intratumoral balance of effector and regulatory T cells resulting in potent anti-tumor efficacy against CT26-HER-2 tumors. Our findings suggest that MVA-BN
®-HER2 generated an anti-tumor response in line with the current paradigm of an “immune signature” [
26] that could lead to protective immunity in humans and warrants further testing of MVA-BN
®-HER2 in clinical trials.
Discussion
Successful cancer immunotherapy has to accomplish two goals: first, the induction of an immune response which is capable of killing cancerous cells; second, to combat or reverse the inflammatory/immunosuppressive mechanisms exerted by the growing tumor that lead to subsequent down-regulation of therapy-induced responses. Pertaining to this challenge, the anti-tumor activity of MVA-BN
®-HER2 was evaluated in a pre-clinical tumor model known to create a strong immunosuppressive environment able to restrain T-cell responses to both tumor-specific and bystander antigens [
23‐
25]. Notably, a single treatment with MVA-BN
®-HER2 showed potent anti-tumor activity against CT26-HER-2 lung metastasis despite the unfavorable immunosuppressive environment. The comparison of HER-2-specific humoral and cellular immune responses induced by treatment of mice with MVA-BN
®-HER2 or HER2+CFA demonstrated that the MVA-BN
® vector exerted potent Th-1 adjuvant activities. More importantly, the quality of the immune response is a crucial factor in cancer immunotherapy as only treatment with MVA-BN
®-HER2 resulted in protective anti-tumor immunity in this aggressive tumor model, while treatment with HER2+CFA did not. Anti-tumor activity was accompanied by a high density of activated CD8
+ T cells along with a clear reduction in the percentage of T
reg cells in the lungs of animals treated with MVA-BN
®-HER2, thus resulting in a crucial shift in the balance of T
eff to T
reg cells in favor of effector T cells [
22]. Importantly, the tumor-infiltrating HER-2-specific CD8 T
eff cells were functional as demonstrated by in vivo CTL activity and intracellular IFN-γ production. These cells were found in the spleen in MVA-BN
®-HER2-treated non-challenged animals and homed to the lungs in tumor-challenged mice. Immunotherapy with MVA-BN
®-HER2 also showed a significant anti-tumor efficacy in a solid CT26-HER2 tumor model (see Supplemental Fig. 3). In this model, epitope spreading to other tumor-associated antigens and long-lasting CTL memory was demonstrated.
A high percentage of CD8 T cells found in lungs of MVA-BN
®-HER2- or MVA-BN
®-treated mice expressed the markers CD11c, NKG2D and KLRG1. The expression of the myeloid marker CD11c on CD8 T cells has been described following acute infections with several viruses including vaccinia [
32]. In general, all CD8
+CD11c
+ T cells also expressed elevated levels of CD44 and have been described in virally infected mice as activated, virus-specific, cytotoxic T cells that are much more effective at controlling viral infections than CD8
+ T cells negative for CD11c [
32‐
34]. Interestingly, CD8
+CD11c
+ T cells have also been identified as effectors of anti-4-1BB-mediated tumor suppression in several mouse tumor immunotherapy models using agonistic anti-4-1-BB antibodies [
35‐
37]. Similar to the results described here, both NKG2D and KLRG-1 were highly expressed on anti-4-1-BB-induced CD8
+CD11c
+ T cells, and they preferentially accumulated in the tumor tissue. Together, these data suggest that CD8
+CD11c
+ T cells found in tumor-bearing lungs are a mixture of highly activated MVA virus- or tumor-specific CD8
+ effector/memory T cells.
The lung in particular is an immunosuppressive environment in which the constitutive production of IL-10 is necessary to protect the integrity of the delicate and highly vascularized tissue. Growing CT26 tumors enhance this already immunosuppressive environment resulting in the recruitment of CD4
+CD25
+FoxP3
+ T
reg cells [
25]. Natural CD4
+CD25
+ T
reg play a critical role in the progression of a number of cancers [
17] and are believed to play a major part in the failure of many immunotherapies against tumors in mice [
24,
38‐
40] and men [
18]. We found that the presence of CT26-HER-2 tumors in the lung consistently increased the percentage of T
reg cells by approximately threefold. The fact that a single treatment with MVA-BN
®-HER2 significantly reduced the frequency of T
reg cells by up to 70% demonstrates that treatment with MVA-BN
®-HER2 can override the potent effects of CD4
+CD25
+FoxP3
+ T
reg cells in establishing conditions conducive to tumor growth, even in the immunosuppressive environment of the lung. In stark contrast, treatment with HER2+CFA resulted in a further increase in the percentage of T
reg cells in the lung and failed to induce any significant anti-tumor efficacy in this model. In vivo depletion experiments further confirmed that the importance of T
reg cells in this tumor model as depletion of either CD4
+ T cells or CD25
+ T cells resulted in a similar partial reduction in tumor burden. MVA-BN
®-HER2 therapy was necessary for the expansion of p63-specific CD8 T cells in both CD4- and CD25-depleted animals, demonstrating that the tumor alone was not able to induce HER-2-specific T cells even in the absence of T
reg cells. However, phenotypic analysis of lung-infiltrating T cells from mice depleted of either CD25
+ or CD4
+ T cells revealed interesting differences. While CD4 depletion resulted in an increase in CD8 T-cell activation irrespective of treatment with MVA-BN
®-HER2, the depletion of CD25
+ cells only increased CD8 T-cell activation in MVA-BN
®-HER2-treated mice. Although the increased activation of CD8 T cells could be responsible for the reduced tumor burden observed in CD4-depleted mice, it is unlikely that CD8
+ T cells are responsible for the reduced tumor size observed in TBS-treated CD25-depleted mice. This could suggest that the absence of T
reg cells might relieve immunosuppressive mechanisms that allow for the activation of cell types other than CD8 T cells. Two likely candidates are CD4 T cells or innate immune effector cells such as NK cells. We are currently investigating the role of CD4 T cells and innate immune mechanisms in MVA-BN
®-HER2-mediated anti-tumor efficacy.
In cancer immunotherapy, a broad response involving all arms of the immune system is desired. Repeated treatment of mice with MVA-BN
®-HER2 resulted in the induction of robust anti-HER-2 antibody responses (supplemental data Fig. 1). Using trastuzumab as a standard, we determined that these titers correlate with 50–500 μg HER-2-specific antibody/mL serum (data not shown), a level within the range of that found in patients treated with trastuzumab [
41,
42]. Although HER-2-specific antibodies do not seem to play a protective role in this particular mouse tumor model, they can be important in other models (our own unpublished observations) and a role for antibody-mediated rejection of HER-2-positive tumors has been well established by the therapeutic activity of trastuzumab. Several other monoclonal antibodies have also demonstrated clinical effectiveness in a variety of malignancies through mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [
43,
44]. Antibody response induced by MVA-BN
®-HER2 showed a strong Th1 bias with IgG2a to IgG1 ratios 500- to 1,000-fold higher than in mice immunized with HER2+CFA. Since Th1 isotypes are thought to be the most efficient mediators of ADCC and CDC, the humoral responses produced by MVA-BN
®-HER2 treatment are desirable and may provide therapeutic activity in human patients.
In conclusion, the data presented here demonstrate that a single treatment with MVA-BN
®-HER2 was able to simultaneously induce Th1-dominated HER-2-specific immune responses and control tumor-induced immunosuppression resulting in potent anti-tumor efficacy. These preclinical results and the excellent safety profile and immunogenicity of MVA-BN
® even in immune-compromised patients (4–6) provide further support for the development of MVA-BN
®-HER2 for cancer immunotherapy. Notably, the immune responses induced by immunotherapy with MVA-BN
®-HER2 are in line with a recently described distinct “immune signature” necessary for the inhibition of a variety of solid tumors treated with different immunotherapeutic strategies including breast cancer [
26,
45‐
48]. Two hallmark features of this signature are the generation of a tumor-specific Th1 responses and a high density of T cells in the tumor parenchyma. The ability of MVA-BN
®-HER2 to induce HER-2-specific immune responses in humans is currently being assessed in Phase I/II clinical trials in HER-2-positive breast cancer patients (NCT00485277).