Federica Pericle

ORAL LACTOFERRIN INHIBITS GROWTH OF ESTABLISHED TUMORS AND POTENTIATES CONVENTIONAL CHEMOTHERAPY

In this work, we investigated the anticancer activity of orally administered recombinant human lactoferrin (rhLF) alone and in combination with chemotherapy in tumor‐bearing mice. rhLF inhibited the growth of squamous cell carcinoma (O12) tumors in T cell–immunocompromised nu/nu mice by 80% when administered at 1,000 mg/kg (2.9 g/m2) by oral gavage twice daily for 8 days (p < 0.001). Similar activity was observed in syngeneic, immunocompetent BALB/c mice, where orally administered rhLF (1,000 mg/kg, 2.9 g/m2 once daily) halted the growth of mammary adenocarcinoma TUBO. Oral rhLF (200 mg/kg, 0.57 g/m2) was also used alone and in combination with cis‐platinum (5 mg/kg) to treat head‐and‐neck squamous cell carcinoma in a syngeneic murine model. Monotherapy with oral rhLF or cis‐platinum caused 61% or 66% tumor growth inhibition over placebo, respectively. Mice receiving both therapies showed 79% growth inhibition, a statistically significant improvement over each drug alone. We then demonstrated that administration of oral rhLF (300 mg/kg, 0.86 g/m2) to tumor‐bearing or naive mice resulted in (i) significantly increased production of IL‐18 in the intestinal tract, (ii) systemic NK cell activation and (iii) circulating CD8+ T‐cell expansion. These data suggest that oral rhLF is an immunomodulatory agent active against cancer as a single agent and in combination chemotherapy, exerting its systemic effect through stimulation of IL‐18 and other cytokines in the gut enterocytes. rhLF has been administered orally to 211 people without a single serious drug‐related adverse event. Thus, rhLF shows promise as a safe and well‐tolerated novel immunomodulatory anticancer agent.

Tyrphostin AG-490 inhibits cytokine-mediated JAK3/STAT5a/b signal transduction and cellular proliferation of antigen-activated human T cells.

Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase required for T cell development and activated by cytokines that utilize the interleukin‐2 (IL‐2) receptor common gamma chain (γc). Genetic inactivation of JAK3 is manifested as severe combined immunodeficiency disease (SCID) in humans and mice. These findings have suggested that JAK3 represents a pharmacological target to control certain lymphoid‐derived diseases. Here we provide novel evidence that AG‐490 potently inhibits the autokinase activity of JAK3 and tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription 5a and 5b (STAT5a/b). Similar inhibitory effects were observed with other cytokines that use γc. AG‐490 also inhibited IL‐2‐mediated proliferative growth in human T cells with an IC50 = 25 μM that was partially recoverable. Moreover, we demonstrate that this inhibitor prevented tetanus toxoid antigen‐specific T cell proliferation and expansion but failed to block activation of Zap70 or p56Lck after anti‐CD3 stimulation of human T cells. Taken together, these findings suggest that AG‐490 inhibits the JAK3‐mediated Type II signaling pathway but not the T cell receptor‐derived Type I pathway and possesses therapeutic potential for T cell‐derived pathologies such as graft‐versus‐host disease, allergy, and autoimmune disorders. J. Leukoc. Biol. 65: 891–899; 1999.

Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells

Lactoferrin (LF) is an important protein component of the innate immune system that is broadly distributed within the body fluids. LF is endowed with multiple biological activities. Talactoferrin (TLF), a recombinant human LF, is in clinical development as an anticancer agent and is entering Phase III clinical trials. Here, we show that TLF induces the maturation of human dendritic cells (DCs) derived from monocytes. TLF, at physiologically relevant concentrations (100 µg/ml) up‐regulates the expression of human leukocyte antigen (HLA) class II, CD83, CD80, and CD86 costimulatory molecule and CXCR4 and CCR7 chemokine receptors, acting primarily through the p38 MAPK signaling pathway. DCs matured by TLF displayed an enhanced release of IL‐8 and CXCL10, as well as a significantly reduced production of IL‐6, IL‐10, and CCL20. They also display a reduced ability to take up antigen and increased capacity to trigger proliferation and release IFN‐γ in the presence of allogeneic human T cells. TLF‐matured DCs are able to prime naive T cells to respond to KLH antigen and display a significantly increased capacity to present Flu‐MA58–66 peptide to HLA‐A2‐matched T cells. These data suggest that a key immunomodulatory function that may be mediated by TLF is to link the innate with adaptive immunity through DC maturation.—Spadaro, M., Caorsi, C., Ceruti, P., Varadhachary, A., Forni, G., Pericle, F., Giovarelli, M. Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells. FASEB J. 22, 2747–2757 (2008)

Direct killing of interleukin-2-transfected tumor cells by human neutrophils

We have previously established that human polymorphonuclear cells (PMN) express IL‐2Rβ‐ and γ‐chains and that addition of IL‐2 maintains the viability of PMN by preventing these cells from undergoing programmed cell death. The purpose of this study was to examine whether IL‐2‐releasing tumor cells are capable of stimulating PMN tumoricidal activity. We therefore investigated the ability of PMN to kill IL‐2‐transfected tumor cells using normal human PMN directed against the murine mammary adenocarcinoma TS/A engineered to release high amounts of murine IL‐2 (3,600 U, B6) compared with TS/A parental cells and TS/A tumor cells transfected with the neomycin‐resistance (NEO) gene only. The potency of PMN as IL‐2‐induced killer cells was indicated by the low number of cells required for killing (effector cell:target cell ratio 10:1) and the degree of tumor cell lysis (68 ± 10%). Evidence for the role of IL‐2 as a mediator of tumor cytotoxicity by PMN was substantiated by inhibition of tumor killing with anti‐IL‐2 and anti‐IL‐2Rβ monoclonal antibodies (MAbs). Furthermore, in vivo depletion of mature granulocytes using MAb RB6‐8C5 resulted in B6 adenocarcinoma growth, thereby confirming a direct role for IL‐2‐activated PMN in tumor cytolysis. Lastly, we suggest that one possible mechanism involved in IL‐2‐induced PMN cytotoxicity against the B6 clone occurs via the nitric oxide pathway, which could be inhibited upon addition of the arginine analog, NG‐monomethyl‐L‐arginine.

Requirement for IFN-gamma, CD8+ T lymphocytes, and NKT cells in talactoferrin-induced inhibition of neu+ tumors

We have previously shown that talactoferrin-alfa (TLF), a recombinant human lactoferrin, is an immunomodulatory protein that is active against implanted tumors, both as a single agent and in combination with chemotherapy. In this study, we show that talactoferrin is active against autochthonous tumors in a transgenic mouse line, which is more analogous to human cancers, and identify key mechanistic steps involved in the anticancer activity of oral TLF. BALB/c mice transgenic for the rat neu (ErbB2) oncogene (BALB-neuT) treated with oral TLF showed a significant delay in carcinogenesis, with 60% tumor protection relative to vehicle-treated mice at week 21. Oral TLF also showed tumor growth inhibition in wild-type BALB/c mice implanted with neu(+) mammary adenocarcinoma, with one third displaying a long-lasting or complete response. Oral TLF induces an increase in intestinal mucosal IFN-gamma production and an increase in Peyers patch cellularity, including expansion of CD8(+) T lymphocytes and NKT cells, and the enhancement of CD8(+) T-cell cytotoxicity. In IFN-gamma knockout mice, there is an absence of the TLF-induced Peyers patch cellularity, no expansion of CD8(+) T lymphocytes and NKT cells, and loss of TLF anticancer activity. TLF antitumor activity is also lost in mice depleted of CD8(+) T cells and in CD1 knockout mice, which lack NKT activity. Thus, the inhibition of distant tumors by oral TLF seems to be mediated by an IFN-gamma-dependent enhancement of CD8(+) T- and NKT cell activity initiated within the intestinal mucosa.

Fusogenic activity of vesicular stomatitis virus glycoprotein plasmid in tumors as an enhancer of IL-12 gene therapy.

We have characterized the fusogenic activity of a plasmid expression system encoding vesicular stomatitis virus G protein (VSVG) in vitro and in vivo. Over 70% of murine colon and renal carcinoma cells (MC38 and Renca, respectively) transfected with VSVG plasmid in vitro fused and formed polykaryons upon incubation with pH 5.5 media. Using a plasmid expression system encoding VSVG and bacterial green fluorescent protein (GFP) formulated in a polyvinyl pyrrolidone (PVP) delivery system, diffusion of GFP throughout the VSVG-induced syncytia was shown in vivo in MC38 and Renca tumors. Moreover, tumor-bearing mice showed tumor growth inhibition following in vivo transfection with VSVG plasmid at an optimal dose of 48 μg. We have previously shown that direct injection of interleukin-12 (IL-12) plasmid complexed with PVP into tumors induces a strong immune response. In the current study, we assessed the ability of VSVG to elicit an antitumor response by enhancing cytokine gene delivery within the tumor mass. Tumor-bearing mice treated intratumorally with both VSVG/PVP and IL-12/PVP (48 and 24 μg, respectively) showed increase in tumor rejection when compared to IL-12 plasmid alone (75% vs. 50%, respectively). These data suggest that VSVG gene therapy can be used in combination with other therapeutic genes to induce an antitumor response in vivo by enhancing the expression of the gene of interest. Cancer Gene Therapy (2001) 8, 55–62

Inhibition of JAK3 with a novel, selective and orally active small molecule induces therapeutic response in T-cell malignancies

Inhibition of JAK3 with a novel, selective and orally active small molecule induces therapeutic response in T-cell malignancies

CD40-CD40L interactions provide "third-party" costimulation for T cell response against B7-1-transfected human breast tumor cells.

In this study we provide evidence that a human breast carcinoma cell line, MDA‐MB‐231 (MDA), can be made immunogenic following B7 transfection and that fall T cell activation is obtained through cooperation of T‐B lymphocytes via CD40‐CD40L interactions. Tumor cells transfected with either B7 gene (MDAB7), neomycin‐resistant gene only (MDAneo), or untransfected (MDA) were used in an allogeneic mixed lymphocyte tumor culture (MLTC) to investigate their ability to stimulate T cell proliferation and generate cytotoxic T lymphocytes (CTL). MDAB7 induced moderate T cell proliferation while MDAneo or MDA did not. Substantial T cell proliferation and de novo generation of cytolytic T cells was obtained only in response to MDAB7 when B cells were present during the MLTC. CD8+‐purified T + B cells proliferated to a greater extent than whole T cell populations + B or CD4+ + B in response to MDAB7. Addition of (α‐B7‐1 or α‐CD40 in the MLTC inhibited T cell proliferation by 65 and 40%, respectively, whereas T cell proliferation and generation of CTL was completely abrogated when MLTC was performed in the presence of both antibodies. These data suggest that the engagement of CD40L on T cells with CD40 on B cells provides a costimulatory signal which, in synergism with TCR‐dependent MDAB7‐T cell recognition (signal 1) and B7/CD28 interactions (signal 2), leads to full T cell activation. J. Leukoc. Biol. 61: 201–208; 1997.

Fighting breast cancer stem cells through the immune-targeting of the xCT cystine–glutamate antiporter

Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2+ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.

Abstract 5572: AX09: an immunotherapy candidate targeting the breast cancer stem cell protein xCT

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks the estrogen, progesterone and HER2 receptors, and accounts for 15-20% of all breast cancers in the US. The particularly aggressive features of TNBC may be due to the enrichment of breast cancer stem cells (BCSC). Due to their resistance to traditional radio- and chemo-therapies, BCSC represent a reservoir for the relapse, metastatic evolution and progression of the disease after treatment. Therefore, successful eradication of BCSC represents a major barrier towards effective cancer treatments. The ability of BCSC to resist common cytotoxic therapies relies on different mechanisms, including improved detoxification ability. The cystine-glutamate antiporter protein xCT (SLC7A11) regulates cystine intake, conversion to cysteine and subsequent glutathione synthesis, protecting cells against oxidative and chemical insults via the p38MAPK pathway. xCT expression is highly restricted to a few normal cell types but is upregulated in a variety of breast cancer subtypes where its expression correlates with poor prognosis. xCT is highly expressed in a variety of solid tumor CSC including BCSC where it interacts with CD44 and plays a functional role in BCSC biology. Agilvax has developed a novel immunotherapy candidate (AX09) based on our virus-like-particle technology for the treatment and prevention of metastatic breast cancer that targets the BCSC protein xCT. Immunization with AX09 elicited a strong antibody response against xCT including high levels of IgG2a antibody. Immune sera from AX09 mice bound to tumorsphere derived BCSC and impacted BCSC function and biology in vitro. To assess if AX09 immunization would decrease metastases, we employed a syngeneic transplantation model, in which purified BCSC derived from TUBO cells were injected into the tail vein of vaccinated female BALB/c mice. Multiple independent experiments showed that immunization with AX09 conferred a significant reduction in the number of pulmonary metastases compared to vaccination with control VLP alone. In a pilot study, BCSC were transplanted into the mammary fat pad and mice were treated with AX09 after primary tumors were 1.5 mm in diameter. Results indicate that AX09 immunization conferred a reduction in lung metastases compared to controls. These data show that an active immunization approach targeting xCT can significantly reduce metastatic progression in preclinical models. Ongoing experiments are further characterizing therapeutic mechanisms and evaluating efficacy of AX09 in combination with front line chemotherapy and checkpoint inhibitors. Citation Format: John O9Rourke, Elisabett Bolli, Valeria Rolih, Laura Conti, Stefania Lanzardo, Jayne Christen, Federica Pericle, Federica Cavallo. AX09: an immunotherapy candidate targeting the breast cancer stem cell protein xCT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5572. doi:10.1158/1538-7445.AM2017-5572