Tumor reductive therapies and antitumor immunity.

Tumor reductive therapy is to reduce tumor burden through direct killing of tumor cells. So far, there is no report on the connection between antitumor immunity and tumor reductive therapies. In the last few years, a new category of cancer treatment, immunotherapy, emerged and they are categorized separately from classic cytotoxic treatments (chemo and radiation therapy). The most prominent examples include cellular therapies (LAK and CAR-T) and immune checkpoint inhibitors (anti-PD-1 and CTLA-4). Recent advances in clinical immunotherapy and our understanding of the mechanism behind them revealed that these therapies have a closer relationship with classic cancer treatments than we thought. In many cases, the effectiveness of classic therapies is heavily influenced by the status of the underlying antitumor-immunity. On the other hand, immunotherapies have shown better outcome when combined with tumor reductive therapies, not only due to the combined effects of tumor killing by each therapy but also because of a synergy between the two. Many clinical observations can be explained once we start to look at these classic therapies from an immunity standpoint. We have seen their direct effect on tumor antigen in vivo that they impact antitumor immunity more than we have realized. In turn, antitumor immunity contributes to tumor control and destruction as well. This review will take the immunological view of the classic therapies and summarize historical as well as recent findings in animal and clinical studies to make the argument that most of the cancer treatments exert their ultimate efficacy through antitumor immunity.

In situ vaccine, immunological memory and cancer cure.

As surgery is able to remove primary tumors and limit metastases, the major challenge in cancer management is the prevention of post-resection recurrence and metastases. From the immune point of view, tumor resection removes the supply of tumor antigens that maintain an active concomitant antitumor immunity elicited by the primary tumor, and may also signal for deposition of immunological memory against future metastases. However, the natural course of this antitumor immunity in many cancer patients following complete tumor resection may not be favorable because protection is often lost after 1-3 years. Recent studies suggest that chemotherapy is able to activate this pre-existing antitumor immunity, and tumor resection following immune activation may lead to higher levels of immunological memory against future tumor antigens (in the form of metastases). Interleukin-12 added to chemotherapy mimics the function of a vaccine adjuvant in that it helps to enhance the antitumor immunity activated by chemotherapy and leaves a much stronger antitumor immune memory. This finding, when applied to cancer management, may help to maintain a strong and long lasting antitumor immunity following complete tumor resection, thus eliminating post-surgery recurrence and metastases.

IL-12 augments antitumor responses to cycled chemotherapy.

Loss of antitumor response to repeated chemotherapy is a major cause of treatment failure in cancer patients. The development of acquired drug resistance is thought to come primarily from changes in tumor cells, and not host response to the tumor. Our recent study shows that antitumor immunity is activated and contributes significantly to the efficacy of chemotherapy. In this study of mouse tumor models, we demonstrate that loss of antitumor response during multiple cycles of chemotherapy is associated with a lack of immune activation, and not intrinsic tumor cell drug resistance. More importantly, we show that adding interleukin-12 (IL-12) to cycled chemotherapy maintains and even increases antitumor immune response in both immunogenic and nonimmunogenic murine tumors and significantly prolongs survival. In some instances, larger tumor burdens that relapse following an initial cycle of cyclophosphamide and IL-12 are eradicated by subsequent cycles of the same treatment at the same doses. Further analysis demonstrates that the initial cycle of the combined therapy increases antitumor immunity of the host. In other mice when tumors are not eradicated by the current cycle of therapy, it serves as a starting point for the subsequent cycles of treatment to generate higher levels of antitumor immunity and greater antitumor response. These results show that the status of host antitumor immunity is a critical factor affecting antitumor efficacy during repeated administration of chemotherapy. Further, IL-12 augments the antitumor immune response under such conditions.

Preexisting antitumor immunity augments the antitumor effects of chemotherapy.

Efficacy of cancer chemotherapy is generally believed to be the result of direct drug killing of tumor cells. However, increased tumor cell killing does not always lead to improved efficacy. Herein, we demonstrate that the status of antitumor immunity at the time of chemotherapy treatment is a critical factor affecting the therapeutic outcome in that tumor-bearing mice that possess preexisting antitumor immunity respond to chemotherapy much better than those that do not. Enhancing antitumor immunity before or at the time of chemotherapy-induced antigen release increases subsequent response to chemotherapy significantly. By in vitro and in vivo measurements of antitumor immunity, we found a close correlation between the intensity of antitumor immunity activated by chemotherapy and the efficacy of treatment. Immune intervention with interleukin-12 during the early phase of chemotherapy-induced immune activation greatly amplifies the antitumor response, often resulting in complete tumor eradication not only at the chemo-treated local site, but also systemically. These findings provide additional evidence for an immune-mediated antitumor response to chemotherapy. Further, our results show that timely immune modification of chemotherapy-activated antitumor immunity can result in enhanced antitumor-immune response and complete tumor eradication.

Lessons from Coley's Toxin.

The active molecule in Coley's Toxin is not tumor necrosis factor (TNF) or endotoxin (LPS), but interleukin-12 (IL-12). IL-12 holds the key to improved anti-tumor immuns response.

Inhibition of host signal transducer and activator of transcription factor 6 results in cure with cyclophosphamide and interleukin 12 immunotherapy.

BACKGROUND: Interleukin (IL)-12 immunotherapy is highly effective against established immunogenic tumors. However, nonimmunogenic tumors fail to respond to IL-12 therapy. Analysis of tumor rejection of the immunogenic tumors shows that a preexisting antitumor immune response is required for an effective IL-12 response. It is not known whether this lack of a preexisting host antitumor immune response is a limiting factor for the lack of response to IL-12 therapy by nonimmunogenic tumors. METHODS: Experiments were done using the spontaneously arising nonimmunogenic metastatic murine breast 4T1 carcinoma in normal and STAT6 knockout BALB/c mice. RESULTS: 4T1 is nonimmunogenic in normal mice, and established subcutaneous tumors are resistant to immunotherapy with cyclophosphamide (Cy) plus IL-12. However, in STAT6 knockout mice, 4T1 becomes immunogenic, and established 4T1 tumors are eradicated by Cy plus IL-12. Adoptive transfer of spleen cells from normal mice into STAT6 knockout mice before tumor inoculation reduces both the immunogenicity and response to Cy plus IL-12 immunotherapy of 4T1 in the recipient mice. CONCLUSIONS: Cy plus IL-12 immunotherapy can eradicate nonimmunogenic tumors as long as a preexisting immunity is established in the tumor-bearing host. Furthermore, the STAT6 pathway is likely involved in the suppression of the development of host antitumor immunity.

Antitumor immunity induced by dendritic cell-based vaccination is dependent on interferon-gamma and interleukin-12.

BACKGROUND: This study was conducted to determine whether dendritic cells (DCs) pulsed with a tumor cell lysate can effectively vaccinate against tumor cells and to establish which cytokines are necessary. MATERIALS AND METHODS: Each wild-type mouse received two subcutaneous immunizations (days 14 and 7) with either saline, tumor lysate, DCs, or tumor-lysate-pulsed DCs. Gamma-interferon (gamma-IFN), knock-out (KO), and interleukin-12 (IL-12) KO mice were also used in immunizations. A tumor challenge was given at day 0. Splenocytes were assayed for gamma-IFN production. RESULTS: All saline-injected mice (n = 19) and all mice injected with tumor lysate (n = 9) developed tumors. Six of nine mice immunized with DCs alone and 6/24 mice treated with lysate-pulsed DCs developed a tumor. Splenocytes from both the saline- and lysate-immunized groups produced undetectable levels of gamma-IFN, while those from mice immunized with either DCs or pulsed DCs produced high levels of gamma-IFN. Four of five gamma-IFN KO mice developed tumors after immunization with tumor-lysate-pulsed DCs. None of four IL-12 KO mice developed a tumor after immunization with wild-type pulsed DCs and 1/10 wild-type mice developed tumor after immunization with IL-12 KO pulsed DCs. Three of four IL-12 KO mice developed tumors after immunization with IL-12 KO pulsed DCs. CONCLUSIONS: Tumor-lysate-pulsed DCs can initiate an effective antitumor immune response. The presence of gamma-IFN in the host is essential for antitumor protection. In contrast, tumor protection is observed if IL-12 is present in either the host or the DCs.

Cure of an established nonimmunogenic tumor, SCC VII, with a novel interleukin 12-based immunotherapy regimen in C3H mice.

OBJECTIVE: To develop a murine model of effective treatment with immunotherapy for established head and neck squamous cell carcinoma. DESIGN: Prospective animal study. Subjects Female C3H mice, 8 to 12 weeks old. INTERVENTIONS: A subcutaneous inoculation of 2 x 10(5) SCC VII cells in C3H mice was established for 7 to 12 days. Tests for concomitant immunity were performed, with and without interleukin 12 modification. Tumors were also tested for responsiveness to interleukin 12 (5 mice) and to cyclophosphamide followed by interleukin 12 (5 mice). SCC VII tumors in 24 mice were treated with interleukin 12 followed by cyclophosphamide and interleukin 12. Five mice with tumors treated with isotonic sodium chloride solution served as controls. Tumors were measured 3 to 4 times weekly, and cure was defined as complete regression of the tumor for at least 60 days. Cured mice were rechallenged with 2 x 10(5) SCC VII cells to verify antitumor immunity. Immunohistochemistry of regressing tumors was performed for CD4+ and CD8+ T cells. RESULTS: Tumor-bearing mice easily developed second tumors when challenged with 2 x 10(5) tumor cells in the opposite flank. However, interleukin 12 treatment provided immunity to second tumors in 8 (100%) of 8 mice when started at day 4 and in 2 (40%) of 5 when treated from day 7. SCC VII did not respond to standard interleukin 12 or cyclophosphamide plus interleukin 12 therapy. Seventy-five percent of animals (18/24) treated with interleukin 12 followed by cyclophosphamide plus interleukin 12 were successfully cured, and all cured mice resisted subsequent challenge with SCC VII. Immunohistochemistry of regressed tumors showed an intense CD4+ and CD8+ infiltrate that was absent in the untreated and nonresponding tumors. CONCLUSIONS: Nonimmunogenic SCC VII is a nonimmunogenic tumor that can be converted into an immunogenic tumor with interleukin 12 treatment. Additional treatment with cyclophosphamide plus interleukin 12 leads to complete regression in 75% of mice.

Production of interferon-gamma by tumor-sensitized T cells is essential for interleukin-12-induced complete tumor eradication.

BACKGROUND: Interferon-gamma (IFN-gamma) is essential for eradication of established large tumors by interleukin-12 (IL-12), but the critical source of IFN-gamma has not been defined. Adoptive transfer of T cells into T cell-deficient mice allows for evaluation of the role of T cells and T cell production of IFN-gamma in the antitumor immune response. METHODS: Wild-type C57BL/6, IL-12 receptor-beta1 knockout (IL-12Rbeta1 KO), IFN-gamma knockout (IFN-gamma KO), and IFN-gamma receptor-alpha knockout (IFN-gammaRalpha KO) mice were immunized and used as donors for adoptive transfer. Transfer of either splenocytes or CD90(+) T cells was performed into recipient T cell receptor-beta knockout (TCRbeta KO) and IFN-gamma/TCRbeta double knockout mice bearing 14-day subcutaneous MCA207 tumors. Half of the mice were treated with IL-12, and cure rates were compared. RESULTS: Transfer of either 1/4 immunized spleen equivalent or 10(7) immunized T cells into both TCRbeta KO and IFN-gamma/TCRbeta KO mice resulted in 80% to 100% cure when given with IL-12. However, transfer of 10(7) immunized T cells from IFN-gamma KO mice into TCRbeta KO mice was ineffective with or without IL-12. T cell response to IL-12, but not IFN-gamma, was required for tumor regression. CONCLUSIONS: Production of IFN-gamma by IL-12-responsive tumor-sensitized T cells is both necessary and sufficient for complete tumor eradication induced by IL-12. T cells are the source, but not the target, of IFN-gamma during tumor regression.

Macrophages as effector cells in interleukin 12-induced T cell-dependent tumor rejection.

Interleukin (IL)-12 activates a T-cell-dependent antitumor immune response that is able to eradicate established large tumors in a number of immunogenic tumor models. The effector mechanisms in these dramatic antitumor responses have not yet been identified. In this report, we show that the effector mechanism of IL-12-induced rejection of established MCA207 tumors is unique in that it is not dependent on perforin, Fas/Fas ligand, and nitric oxide. Study of cyclophosphamide plus IL-12 (Cy + IL-12)-induced rejection of ascites Sa1 tumor demonstrates that macrophages are the predominant immune cell infiltration in the ascites. These macrophages possess nonspecific tumoricidal activity in vivo as immune distinct MCA207 tumor cells inoculated i.p., but not s.c., in mice bearing regressing Sa1 ascites tumors after Cy + IL-12 therapy are rejected. Furthermore, Cy + IL-12-treated Sa1 ascites cells or macrophages, but not spleen macrophages from the same mouse or inflammatory macrophages induced by thioglycollate, are able to suppress the development of immune-irrelevant s.c. tumors in a Winn assay. These macrophages kill various tumor cells in a contact-dependent manner in vitro, and the cytotoxicity is preserved after fixation with paraformaldehyde. These results demonstrate that activated macrophages function as effector cells in an IL-12-induced, T-cell-dependent eradication of established tumors through a novel contact-dependent, paraformaldehyde fixation-resistant, apoptosis-inducing mechanism.

The role of IFN-gamma in rejection of established tumors by IL-12 : source of production and target.

We have demonstrated previously that established small and large murine MCA207 sarcomas can be completely eradicated by treatment with interleukin (IL) 12 alone and cyclophosphamide plus IL-12 (Cy+IL-12), respectively. The antitumor effect of IL-12/Cy+IL-12 has been found to be dependent on IFN-gamma and T cells. The role of IFN-gamma in IL-12-induced tumor rejection is unclear, because after IL-12 administration IFN-gamma is produced by multiple cell types, and it acts on most cell types because of the ubiquitous expression of its receptor. Using a T-cell-adoptive transfer model, we show that after IL-12 treatment, tumor-specific T-cell production of IFN-gamma is necessary and sufficient for rejection of established tumors. Furthermore, by testing tumors using IFN-gamma-unresponsive tumor cells, we show that tumor cell expression of MHC class I molecules in vivo is abrogated by blocking the response to IFN-gamma. However, tumor response to IFN-gamma is not essential for rejection of established small and large tumors by IL-12 and Cy+IL-12, respectively; neither is it essential for expression of tumor immunogenicity. Our results indicate that the rejection of established tumors by IL-12/Cy+IL-12 is dependent on the induction of a Th1 response producing IFN-gamma that acts on host cells.