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Background: The efficacy of PD-(L)1 inhibitors in patients with trastuzumab-resistant advanced HER2+ breast cancer is poor. Although many HER2 targeted therapies are used clinically, their effect on the tumor immune microenvironment (TME) and whether this contributes to efficacy is not understood. Tucatinib is a potent, highly selective, HER2 small molecule tyrosine kinase inhibitor with proven clinical benefit in the advanced setting. Methods: We used two immunocompetent, HER2+ murine cancer models (trastuzumab-sensitive H2N113 and trastuzumab-resistant fo5) to investigate the effects of tucatinib on tumor growth kinetics, as well as tucatinib anti-tumor efficacy in combination with trastuzumab and PD-1 checkpoint blockade. Effects of tucatinib on the tumor infiltrating lymphocytes were analysed using flow cytometry. To identify genes that were modulated by tucatinib treatment, we performed bulk RNA sequencing on fo5 tumors treated with vehicle or tucatinib. We evaluated healthy donor peripheral blood T cells treated with tucatinib for 96 hours and analysed secreted cytokine levels using cytometric bead array. Results: Treatment of CD4+ and CD8+ T cells with tucatinib together with TCR stimulation resulted significantly higher levels of IFNγ and TNFα compared with stimulated controls (p < 0.0001), suggesting tucatinib can directly modulate human T cell function. In both murine models, tucatinib significantly inhibited tumor growth in a dose-dependent manner and was observed at doses of 25 mg/kg, 50 mg/kg and 100 mg/kg (p < 0.05 between each dose). Median survival was 16 days in the vehicle group vs 50 days with 100 mg/kg of tucatinib (p < 0.0001). Ex vivo analysis of tumors by flow cytometry showed increased infiltration of NK cells (p=0.01) and, CD8+ T cells with high PD-1 (p = 0.001), TIM-3 (p = 0.009), IFNγ (p = 0.003) and Ki67 (p = 0.0003) expression in Tucatinib treated mice compared to vehicle treated mice. Concomitant with these changes there was a significant reduction in numbers of neutrophils (p = 0.0085) and MHC-II low expressing macrophage populations (p < 0.0001) following tucatininb treatment with an increase in frequency of MHC-II expressing dendritic cells (p < 0.0001) and macrophages (p < 0.0001) suggesting an increase in anti-tumor immunity. Similarly, in the fo5 model tucatinib treated tumors had significantly higher interferon-γ (IFNγ) produced by CD8+ T cells (p = 0.005). Gene expression profile analysis shows significant enrichment in pathways associated with immune activation, including antigen binding and presentation (p = 0.0002), adaptive immune responses (p = 0.0002) including IFNγ (p = 0.0002) and IFNα (p = 0.0002). In this model, tucatinib in combination with trastuzumab demonstrated significantly better anti-tumor activity (p = 0.03) and survival (p < 0.0001) compared with tucatinib alone, with 33% of mice achieving complete tumour regressions. Tucatinib in combination with PD-1 inhibition also demonstrated significantly greater anti-tumor efficacy compared to tucatinib alone (p = 0.0079) with increased survival (p = 0.05) and 50% of mice achieving complete tumor regression. Conclusions: This study suggests an anti-tumor immune response may be an important component of the efficacy of tucatinib. This is supported by in vitro data demonstrating tucatinib stimulated human peripheral T cells and in vivo data showing favorable effects on the TME mediated by tucatinib treatment. These changes were associated with improved efficacy when tucatinib was combined with PD-1 inhibition or trastuzumab in the setting of trastuzumab resistance. These findings suggest that the combination of tucatinib and PD-1 inhibition is a rational combination that warrants investigation in the clinical setting, particularly for trastuzumab resistant HER2+ breast tumors. Citation Format: Ran Li, Sneha Sant, Emmaline Brown, Franco Caramia, Ann Byrne, Kylie Clarke, Michael Neeson, Paul J Neeson, Phillip K Darcy, Scott Peterson, Sherene Loi. Tucatinib favourably modulates the immune microenvironment and synergises with anti-PD1 therapy in a trastuzumab resistant HER2+ murine model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-04.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296–309. ©2017 AACR.

Objective: The aim is to demonstrate dynamic in-vivo tracking of CAR T cell therapy for treatment of solid tumors using Cu-64 labeled superparamagnetic iron oxide nanoparticles (SPION) as novel dual PET-MR imaging agent. Methodology: Cu-64 SPION: Cu-64 radioisotope is bound to silica coated SPION using enhanced electrolysis plating techniques with tin and palladium seeding. Preclinical Model: Mouse splenic T cells were activated with anti-CD3, anti-CD28 & cultured with IL-2 and IL-7, prior to being transduced with second generation anti-Her-2 CAR (scFv-CD28-CD3ζ). 5 x 105 E0771-hHER2 breast tumor cells were implanted subcutaneously into male C57Bl/6-human HER2 transgenic mice. 107 labeled CAR T or control T cells (Cu-64 5-8 MBq) were injected into tail vein. Clinical Model: Activated T cells using antibody CD3 (OKT3) & IL-2 are transduced with retroviral vector constructs encoding for chimeric T-cell receptor specific for Lewis Y antigen. Modified T-cells are further expanded ex-vivo and reinfused. 3 x 108 CAR T cells were labeled with Cu-64 (200 - 300 MBq). Labeling of CAR T cells with Cu-64 SPION: Transfecting agent protamine sulphate facilitated cellular uptake of Cu-64 SPION within cells. Functional assays: 51Chromium release, cytometric bead array and cell viability showed that labeling process did not affect CAR T cell cytotoxicity, cytokine secretion (TNFα and IFN-γ) and viability. CAR T Cell Tracking: Scanning was performed using clinical grade dual PET-MR scanner. Preliminary Data: In this clinical trial (HREC/16/PMCC/30) patients are being enrolled for first in human in vivo study to determine how many cells distribute to solid tumor sites within first few days of CAR T cell therapy. This is first data that demonstrates that CAR-T cells can be consistently and efficiently labeled (≤60%) with cell viability (≥85%) and at sensitivity comparable to detecting at least z cells at tumor site using clinical grade dual PET-MR scanner. SUVmean values provides insight into individual response to therapy. The observed increase in SUVmax over time specifies localization of CAR T cells at tumor sites. Clinical data at early time point showed moderate uptake in lungs posterior basal segments without increased activity over next few days, thus suggesting transient process. Mild, diffuse bone marrow and relatively intense uptake in the liver and spleen suggests margination of cells to the reticulo-endothelial system. Distinct PET signal suggests localization of labeled cells in the secondary tumor sites. Little background uptake in important organs such as brain and heart indicate the safety profile of imaging agent. Absence of signal in bladder indicates hepatobiliary excretion, which may allow re-absorption from GI tract and re-circulation. Distinct PET signal within tumor in preclinical studies confirms trafficking of CAR T cells to tumor site as compared to controls. A negative contrast in the liver on T2 weighted MRI in both the preclinical and clinical studies. Preliminary Conclusion:This is first in human in vivo study to show CAR T cell distribution in real time and provides insight into individual responses of tumors to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The preliminary findings indicate that labeled cells traffic to tumor sites in first few hours of infusion and remain persistent for extended period. Citation Format: Ritu Singla, Dominic Wall, Samuel Anderson, Nicholas Zia, James C. Korte, Lucy Kravets, Gerard McKiernan, Jeanne Butler, Amanda Gammilonghi, Jyoti Arora, Ben Solomon, Rodney Hicks, Timothy Cain, Phillip Darcy, Carleen Cullinane, Paul Neeson, Rajesh Ramanathan, Ravi Shukla, Vipul Bansal, Simon Harrison. Dynamic real time in vivo CAR T cell tracking: Clinical and preclinical studies using a novel dual PET-MR imaging agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-023.

New treatments for triple-negative breast cancer (TNBC) are urgently needed. Despite there being little evidence of clinical activity as single-agent therapies, we show that dual blockade of PI3Kα and CDK4/6 is synergistically effective against multiple RB1-wild-type TNBC models. Combined PI3Kα and CDK4/6 inhibition significantly increased apoptosis, cell-cycle arrest, and tumor immunogenicity and generated immunogenic cell death in human TNBC cell lines. Combination treatment also significantly improved disease control in human xenograft models compared with either monotherapy. Combined PI3Kα and CDK4/6 inhibition significantly increased tumor-infiltrating T-cell activation and cytotoxicity and decreased the frequency of immunosuppressive myeloid-derived suppressor cells in a syngeneic TNBC mouse model. Notably, combined PI3Kα and CDK4/6 inhibition, along with inhibition of immune checkpoints PD-1 and CTLA-4, induced complete and durable regressions (>1 year) of established TNBC tumors in vivo. Overall, our results illustrate convergent mechanisms of PI3Kα and CDK4/6 blockade on cell-cycle progression, DNA damage response, and immune-modulation and may provide a novel therapeutic approach for TNBC. Cancer Res; 77(22); 6340–52. ©2017 AACR.

Histone deacetylase inhibitors (HDACi) may engage host immunity as one basis for their antitumor effects. Herein, we demonstrate an application of this concept using the HDACi panobinostat to augment the antitumor efficacy of trastuzumab (anti-HER2) therapy, through both tumor cell autonomous and nonautonomous mechanisms. In HER2+ tumors that are inherently sensitive to the cytostatic effects of trastuzumab, cotreatment with panobinostat abrogated AKT signaling and triggered tumor regression in mice that lacked innate and/or adaptive immune effector cells. However, the cooperative ability of panobinostat and trastuzumab to harness host anticancer immune defenses was essential for their curative activity in trastuzumab-refractory HER2+ tumors. In trastuzumab-resistant HER2+ AU565pv xenografts and BT474 tumors expressing constitutively active AKT, panobinostat enhanced the antibody-dependent cell-mediated cytotoxicity function of trastuzumab. IFNγ–mediated, CXCR3-dependent increases in tumor-associated NK cells underpinned the combined curative activity of panobinostat and trastuzumab in these tumors. These data highlight the immune-enhancing effects of panobinostat and provide compelling evidence that this HDACi can license trastuzumab to evoke NK-cell–mediated responses capable of eradicating trastuzumab-refractory HER2+ tumors. Cancer Res; 77(10); 2594–606. ©2017 AACR.

CD73 is a cell surface 5′-nucleotidase that converts AMP to adenosine, an immune suppressive molecule. CD73 may promote immune escape in cancer by contributing to the degradation of extracellular ATP released by dying cancer cells in hypoxic tumors or following chemotherapy. However, whether CD73 exerts a critical oncogenic function during tumorigenesis is unknown. In this study, we used genetically deficient mice to investigate its contribution to autochthonous tumor formation. CD73 deficiency suppressed the development of 3-methylcholanthrene (MCA)-induced fibrosarcomas through a mechanism relying upon IFN-γ, natural killer (NK) cells, and CD8+ T cells. Similarly, CD73 deficiency also suppressed prostate tumorigenesis in TRAMP transgenic mice. Importantly, treatment with an anti-CD73 monoclonal antibody effectively suppressed growth of established MCA-induced tumors or TRAMP-C1 prostate tumors and inhibited the development of TRAMP-C1 lung metastases. The therapeutic activity of anti-CD73 monoclonal antibody against primary tumors was dependent on CD8+ T cells, whereas its antimetastatic activity was dependent on host CD73 expression independent of T cells or NK cells. Taken together, our findings indicate that CD73 is a critical factor in tumorigenesis and that anti-CD73 antibodies may offer a novel generalized strategy to blunt immune escape and treat cancer. Cancer Res; 72(9); 2190–6. ©2012 AACR.

CD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on antitumor immunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. The protective effect of CD73 deficiency on primary tumors was dependent on CD8+ T cells and associated with an increased frequency of antigen-specific CD8+ T cells in peripheral blood and tumors and increased antigen-specific IFN-γ production. Replicate studies in bone marrow chimeras established that both hematopoietic and nonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptive reconstitution of T regulatory cell (Treg)–depleted DEREG (depletion of regulatory T cells) mice, we demonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73. CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells after intravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, most likely endothelial cells, was critical for promoting lung metastasis in a manner independent from immunosuppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonal antibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, our findings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the level of tumor cells, Tregs, and nonhematopoietic cells. Cancer Res; 71(8); 2892–900. ©2011 AACR.

Acting as both a polarity regulator and a scaffolding protein, Scribble (SCRIB), coordinates intracellular signalling pathways that regulate cell proliferation, migration and apoptosis. Deregulation of SCRIB has been linked to multiple epithelial cancers but the role of SCRIB in hematopoiesis and hematopoietic malignancies remains relatively unexplored. Using different approaches, we show that SCRIB is expressed in hematopoietic stem and progenitor cells and in their differentiated progenies, implying a role for SCRIB in blood production. We investigated the effect of SCRIB loss in hematopoiesis using two conditional knockout mouse models in conjunction with an extensive phenotypic labelling regime and multi-color flow cytometry. We reveal a role for SCRIB in T cell development although subtle differences between the two mouse models were noted. Compensatory mechanisms often mask the effects of single protein deletion during steady-state so stress hematopoiesis was induced through a transplant assay to minimise these effects. Lethally irradiated recipient mice receiving a transplant of SCRIB knockout bone marrow revealed a more striking phenotype with significantly altered populations of granulocyte, macrophage, erythroid, T and B cell progenitors, and mature macrophage fractions compared to controls. Collectively, our data shows SCRIB is required for the development of multiple hematopoietic fractions and studies are currently underway to identify the underlying mechanism/s and elucidate how deregulation of SCRIB impacts on hematopoietic malignancies. Citation Format: Novita Novita, Alysha Dew, Chad Johnson, Carl R. Walkley, Louise E. Purton, Patrick O. Humbert, Phillip K. Darcy, Sarah L. Ellis. Investigating the role of polarity protein, SCRIB, in hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-183.

Adoptive immunotherapy utilising chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematological cancers, with up to 90% complete remission being reported in patients with acute lymphoblastic leukemia (ALL). However, this success is not currently transferrable to solid malignancies due in part to the immunosuppressive tumor microenvironment limiting CAR T cell responses. Given that activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong co-stimulatory signals for augmenting and diversifying T cell responses, we hypothesized that the combination of α-4-1BB and CAR T cell therapy would result in improved anti-tumor responses. Using a human-Her2 self-antigen mouse model, we found that α-4-1BB significantly enhanced CAR T cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Investigation into the mechanism revealed increased expression of IFNγ and the proliferation marker Ki67 by tumor-infiltrating CAR T cells when combined with α-4-1BB. The importance of IFNγ was demonstrated in vivo where synergistic effects of the combination therapy were abrogated when IFNγ was depleted. Strikingly, we also found that α-4-1BB significantly reduced host immunosuppressive cells at the tumor site including regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), correlating with an increased therapeutic response. Investigation in immunocompromised RAG -/- mice showed reduced synergistic effects following the combination therapy, suggesting some contribution by host T cells. Furthermore, we found increased frequency of endogenous T cells and dendritic cells in the tumor-draining lymph nodes, supporting potential engagement of host immune cells. We therefore conclude α-4-1BB has a multifunctional role for enhancing CAR T cell responses including engagement of endogenous immune cells, and this combination approach has high translational potential given that CAR T cell therapy has recently been FDA approved for pediatric ALL, and that α-4-1BB is currently being tested in clinical trials against various types of cancer. Citation Format: Sherly Mardiana, Liza B. John, Michael H. Kershaw, Paul A. Beavis, Phillip K. Darcy. A multifunctional role for adjuvant anti-4-1BB therapy in augmenting antitumor responses by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1530.

Given that specific subsets of T helper 1 (Th1) and T helper 2 (Th2) CD4+ T cells have been shown to play key roles in tumor rejection models, we wanted to assess the contribution of either Th1 or Th2 CD4+ cell subtypes for redirected T-cell immunotherapy. In this study, we have developed a novel method involving retroviral transduction and in vitro T-cell polarization to generate gene-engineered mouse CD4+ Th1 and Th2 cells or T helper intermediate (Thi) cells expressing an anti–erbB2-CD28-ζ chimeric receptor. Gene-modified Th1 and Th2 polarized CD4+ cells were characterized by the preferential secretion of IFN-γ and interleukin-4, respectively, whereas Thi cells secreted both cytokines following receptor ligation. In adoptive transfer studies using an erbB2+ lung metastasis model, complete survival of mice was observed when transduced Th1, Th2, or Thi CD4+ cells were transferred in combination with an equivalent number of transduced CD8+ T cells. Tumor rejection was consistently associated with transduced T cells at the tumor site and interleukin-2 secretion. However, the surviving mice treated with gene-modified Th1 CD4+ cells were significantly more resistant to a subsequent challenge with a different erbB2+ tumor (4T1.2) implanted s.c. This result correlated with both increased expansion of Th1 CD4+ and CD8+ T cells in the blood and a greater number of these cells localizing to the tumor site following rechallenge. These data support the use of gene-modified CD4+ Th1 and CD8+ T cells for mediating a sustained antitumor response. [Cancer Res 2007;67(23):11428–37]

T cells are a crucial component of the immune response to infection and cancer. In addition to coordinating immunity in lymphoid tissue, T cells play a vital role at the disease site, which relies on their efficient and specific trafficking capabilities. The process of T-cell trafficking is highly dynamic, involving a series of distinct processes, which include rolling, adhesion, extravasation, and chemotaxis. Trafficking of T cells to the tumor microenvironment is critical for the success of cancer immunotherapies such as adoptive cellular transfer. Although this approach has achieved some remarkable responses in patients with advanced melanoma and hematologic malignancy, the success against solid cancers has been more moderate. One of the major challenges for adoptive immunotherapy is to be able to effectively target a higher frequency of T cells to the tumor microenvironment, overcoming hurdles associated with immunosuppression and aberrant vasculature. This review summarizes recent advances in our understanding of T-cell migration in solid cancer and immunotherapy based on the adoptive transfer of natural or genetically engineered tumor-specific T cells and discusses new strategies that may enhance the trafficking of these cells, leading to effective eradication of solid cancer and metastases. Cancer Res; 74(24); 7168–74. ©2014 AACR.

Chimeric antigen receptor (CAR) T cell therapy is a novel form of adoptive cellular therapy and has recently generated remarkable effects in patients with hematological cancers. However, the success against solid cancers has been modest. The major challenges are the hostile tumor microenvironment and the low efficiency of CAR T cells infiltrating the tumor. Here, we present a major advancement in CAR T therapy that eradicated large established solid cancers, some in excess of 150 mm2, in immunocompetent mice. We hypothesized that a vaccine composed of a recombinant poxvirus could be used as an antigen delivery vehicle to specifically activate CAR T cells through their T cell receptor (TCR) and simultaneously change the tumor microenvironment, allowing the recruitment and activation of CAR T cells. The approach involves adoptive cell transfer incorporating vaccination (ACTIV) therapy. We generated dual-specific T cells expressing a CAR specific for the tumor antigen Her2 and a TCR specific for the melanocyte protein (gp100). Injection of T cells, together with recombinant vaccinia virus expressing gp100, induced durable complete remission of a variety of Her2+ tumors and established metastases, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Tumor destruction mediated by dual-specific T cells occurred rapidly over a period of seven days and was associated with an extensive proliferation and infiltration of the dual-specific CAR T cells. Mice that had rejected tumors were resistant to rechallenge with the same Her2+ tumor cells and partially resistant to rechallenge with Her2- tumor cells, indicating the formation of immune memory and epitope spreading. This mouse model study supports the view that it is possible to design a highly effective CAR T cell therapy for solid cancers and metastases, even when the target antigen is also expressed in vital tissues. To explore the translational potential for using the dual specific CAR T cell strategy, we established methods to transduce the T cells from human peripheral blood with both a TCR specific for gp100 and a CAR for Her2. From as little as 1 ml of human buffy coat, we could generate more than 109 dual-specific CAR T cells, which is sufficient for a course of treatment. The human dual-specific CAR T cells were functional in secreting IFN-γ and killing human cancer cells when co-cultured with the gp100 or Her2 expressing human cancer cells. The stimulation of gp100 through TCR enhanced the dual-specific CAR T cell proliferation, secretion of IFN-γ and killing of Her2+ human cancer cells in vitro. These characteristics were identified to be important for eradicating tumors in the mouse models. Taken together, our data provide valuable information for the development of CAR T cell therapies for patients with solid cancers. Citation Format: Clare Y. Slaney, Bianca von Scheidt, Phillip K. Darcy, Michael H. Kershaw. Dual-specific T cells are highly effective in eradicating solid tumors [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 631. doi:10.1158/1538-7445.AM2017-631

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b+ and CD11c+ myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8+ effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer. Cancer Res; 72(7); 1651–60. ©2012 AACR.

Adoptive immunotherapy involving genetic modification of T cells with antigen-specific, chimeric, single-chain receptors is a promising approach for the treatment of cancer. To determine whether gene-modified T cells could induce antitumor effects without associated autoimmune pathology, we assessed the ability of T cells expressing an anti-Her-2 chimeric receptor to eradicate tumor in Her-2 transgenic mice that express human Her-2 as a self-antigen in brain and mammary tissues. In adoptive transfer studies, we demonstrated significant improvement in the survival of mice bearing Her-2+ 24JK tumor following administration of anti-Her-2 T cells compared with control T cells. The incorporation of a lymphoablative step prior to adoptive transfer of anti-Her-2 T cells and administration of IL-2 were both found to further enhance survival. The reduction in tumor growth was also correlated with localization of transferred T cells at the tumor site. Furthermore, an antigen-specific recall response could be induced in long-term surviving mice following rechallenge with Her-2+ tumor. Importantly, antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. This study highlights the therapeutic potential of using gene-engineered T cells as a safe and effective treatment of cancer. Cancer Res; 70(23); 9591–8. ©2010 AACR.

Several reports have shown that prophylactic depletion of regulatory T cells (Treg) using various monoclonal antibodies (mAb) in mice can stimulate potent antitumor immune responses and prevent tumor development. These same depletion methods do not significantly suppress tumor growth in a therapeutic setting. Although different strategies to deplete FoxP3+ Treg have been used, no study has systematically compared these qualitatively for the effector mechanisms they each liberate. Herein, using prophylactic depletion of FoxP3+ Tregs with either anti-CD4, anti-CD25, or anti-FR4 mAbs, we have compared the cellular and effector requirements for elimination of the renal carcinoma RENCA and prevention of methylcholanthrene-induced fibrosarcoma. Collectively from these two models, it was clear that CD8+ T cells and natural killer cells played an important role downstream of Treg depletion. However, whereas all three mAbs quantitatively depleted FoxP3+ T cells to a similar extent, subtle differences in the downstream mechanisms of tumor control existed for all three approaches. In general, neutralization of any lymphocyte subset or effector mechanism was insufficient to alter tumor suppression initiated by Treg depletion, and in some settings, the neutralization of multiple effector mechanisms failed to prevent tumor rejection. These studies reveal that Tregs control multiple redundant elements of the immune effector response capable of inhibiting tumor initiation and underscore the importance of effectively targeting these cells in any cancer immunotherapy. Cancer Res; 70(7); 2665–74

A rational monoclonal antibody (mAb)-based antitumor therapy approach has previously been shown to eradicate various established experimental and carcinogen-induced tumors in a majority of mice. This therapy comprised an agonistic mAb reactive with tumor necrosis factor–related apoptosis-inducing ligand receptor (DR5), expressed by tumor cells, an agonistic anti-CD40 mAb to mature dendritic cells, and an agonistic anti-4-1BB mAb to costimulate CD8+ T cells. Because agonists of CD40 have been toxic in patients, we were interested in substituting anti-CD40 mAb with other dendritic cell–maturing agents, such as glycolipid ligands recognized by invariant natural killer T (iNKT) cells. Here, we show that CD1d-restricted glycolipid ligands for iNKT cells effectively substitute for anti-CD40 mAb and reject established experimental mouse breast and renal tumors when used in combination with anti-DR5 and anti-4-1BB mAbs (termed “NKTMab” therapy). NKTMab therapy–induced tumor rejection was dependent on CD4+ and CD8+ T cells, NKT cells, and the cytokine IFN-γ. NKTMab therapy containing either α-galactosylceramide (α-GC) or α-C-galactosylceramide (α-c-GC) at high concentrations induced similar rates of tumor rejection in mice; however, toxicity was observed at the highest doses of α-GC (>250 ng/injection), limiting the use of this glycolipid. By contrast, even very low doses of α-c-GC (25 ng/injection) retained considerable antitumor activity when used in combination with anti-DR5/anti-4-1BB, and thus, α-c-GC showed a considerably greater therapeutic index. In summary, sequential tumor cell apoptosis and amplification of dendritic cell function by NKT cell agonists represents an exciting and novel approach for cancer treatment. [Cancer Res 2007;67(15):7495–504]