Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models.

Prolonged activation of vascular endothelial growth factor receptor-2 (VEGFR-2) due to mis-regulation of the VEGF pathway induces aberrant blood vessel expansion, which supports growth and survival of solid tumors. Therapeutic interventions that inhibit the VEGFR-2 pathway have therefore become a mainstay of cancer treatment. Non-clinical studies have recently revealed that blockade of angiogenesis can modulate the tumor microenvironment and enhance the efficacy of concurrent immune therapies. Ramucirumab is an FDA-approved anti-angiogenic antibody that inhibits VEGFR-2 and is currently being evaluated in clinical studies in combination with anti-programmed cell death (PD-1) axis checkpoint inhibitors (pembrolizumab, durvalumab, or sintilimab) across several cancer types. The purpose of this study is to establish a mechanistic basis for the enhanced activity observed in the combined blockade of VEGFR-2 and PD-1-axis pathways. Pre-clinical studies were conducted in murine tumor models known to be responsive to anti-PD-1 axis therapy, using monoclonal antibodies that block mouse VEGFR-2 and programmed death-ligand 1 (PD-L1). Combination therapy resulted in enhanced anti-tumor activity compared to anti-PD-L1 monotherapy. VEGFR-2 blockade at early timepoints post-anti-PD-L1 therapy resulted in a dose-dependent and transient enhanced infiltration of T cells, and establishment of immunological memory. VEGFR-2 blockade at later timepoints resulted in enhancement of anti-PD-L1-driven immune cell infiltration. VEGFR-2 and PD-L1 monotherapies induced both unique and overlapping patterns of immune gene expression, and combination therapy resulted in an enhanced immune activation signature. Collectively, these results provide new and actionable insights into the mechanisms by which concurrent VEGFR-2 and PD-L1 antibody therapy leads to enhanced anti-tumor efficacy.

Preclinical Evaluation of 89Zr-Df-IAB22M2C PET as an Imaging Biomarker for the Development of the GUCY2C-CD3 Bispecific PF-07062119 as a T Cell Engaging Therapy.

PURPOSE: A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining. PROCEDURES: NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells. RESULTS: The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119. CONCLUSION: Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

Immunomodulatory Activity of a Colony-stimulating Factor-1 Receptor Inhibitor in Patients with Advanced Refractory Breast or Prostate Cancer: A Phase I Study.

PURPOSE: Tumor-associated macrophages correlate with increased invasiveness, growth, and immunosuppression. Activation of the colony-stimulating factor-1 receptor (CSF-1R) results in proliferation, differentiation, and migration of monocytes/macrophages. This phase I study evaluated the immunologic and clinical activity, and safety profile of CSF-1R inhibition with the mAb LY3022855. PATIENTS AND METHODS: Patients with advanced refractory metastatic breast cancer (MBC) or metastatic castration-resistant prostate cancer (mCRPC) were treated with LY3022855 intravenously in 6-week cycles in cohorts: (A) 1.25 mg/kg every 2 weeks (Q2W); (B) 1.0 mg/kg on weeks 1, 2, 4, and 5; (C) 100 mg once weekly; (D)100 mg Q2W. mCRPC patients were enrolled in cohorts A and B; patients with MBC were enrolled in all cohorts. Efficacy was assessed by RECIST and Prostate Cancer Clinical Trials Working Group 2 criteria. RESULTS: Thirty-four patients (22 MBC; 12 mCRPC) received ≥1 dose of LY3022855. At day 8, circulating CSF-1 levels increased and proinflammatory monocytes CD14DIMCD16BRIGHT decreased. Best RECIST response was stable disease in five patients with MBC (23%; duration, 82-302 days) and three patients with mCRPC (25%; duration, 50-124 days). Two patients with MBC (cohort A) had durable stable disease >9 months and a third patient with MBC had palpable reduction in a nontarget neck mass. Immune-related gene activation in tumor biopsies posttreatment was observed. Common any grade treatment-related adverse events were fatigue, decreased appetite, nausea, asymptomatic increased lipase, and creatine phosphokinase. CONCLUSIONS: LY3022855 was well tolerated and showed evidence of immune modulation. Clinically meaningful stable disease >9 months was observed in two patients with MBC.

The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy.

PURPOSE: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non-small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. EXPERIMENTAL DESIGN: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. RESULTS: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. CONCLUSIONS: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890.

Rational design of anti-GITR-based combination immunotherapy.

Modulating T cell homeostatic mechanisms with checkpoint blockade can efficiently promote endogenous anti-tumor T cell responses1-11. However, many patients still do not benefit from checkpoint blockade12, highlighting the need for targeting of alternative immune pathways13. Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is an attractive target for immunotherapy, owing to its capacity to promote effector T cell (Teff) functions14,15 and hamper regulatory T cell (Treg) suppression16-20. On the basis of the potent preclinical anti-tumor activity of agonist anti-GITR antibodies, reported by us and others16,21,22, we initiated the first in-human phase 1 trial of GITR agonism with the anti-GITR antibody TRX518 ( NCT01239134 ). Here, we report the safety profile and immune effects of TRX518 monotherapy in patients with advanced cancer and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. We demonstrate that TRX518 reduces circulating and intratumoral Treg cells to similar extents, providing an easily assessable biomarker of anti-GITR activity. Despite Treg reductions and increased Teff:Treg ratios, substantial clinical responses were not seen. Similarly, in mice with advanced tumors, GITR agonism was not sufficient to activate cytolytic T cells due to persistent exhaustion. We demonstrate that T cell reinvigoration with PD-1 blockade can overcome resistance of advanced tumors to anti-GITR monotherapy. These findings led us to start investigating TRX518 with PD-1 pathway blockade in patients with advanced refractory tumors ( NCT02628574 ).

FcγRIIIa-dependent IFN-γ release in whole blood assay is predictive of therapeutic IgG1 antibodies safety.

Immunomodulatory monoclonal IgG1 antibodies developed for cancer and autoimmune disease have an inherent risk of systemic release of pro-inflammatory cytokines. In vitro cytokine release assays are currently used to predict cytokine release syndrome (CRS) risk, but the validation of these preclinical tools suffers from the limited number of characterized CRS-inducing IgG1 antibodies and the poor understanding of the mechanisms regulating cytokine release. Here, we incubated human whole blood from naïve healthy volunteers with four monoclonal IgG1 antibodies with different proven or predicted capacity to elicit CRS in clinic and measured cytokine release using a multiplex assay. We found that, in contrast to anti-CD52 antibodies (Campath-1H homolog) that elicited high level of multiple inflammatory cytokines from human blood cells in vitro, other IgG1 antibodies with CRS-inducing potential consistently induced release of a single tested cytokine, interferon (IFN)-γ, with a smaller magnitude than Campath. IFN-γ expression was observed as early as 2-4 h after incubation, mediated by natural killer cells, and dependent upon tumor necrosis factor and FcγRIII. Importantly, the magnitude of the IFN-γ response elicited by IgG1 antibodies with CRS-inducing potential was determined by donor FcγRIIIa-V158F polymorphism. Overall, our results highlight the importance of FcγRIIIa-dependent IFN-γ release in preclinical cytokine release assay for the prediction of CRS risk associated with therapeutic IgG1 antibodies.

Targeting the TGFß pathway with galunisertib, a TGFßRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade.

BACKGROUND: TGFß signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFß's immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFß pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. RESULTS: In vitro treatment with galunisertib reversed TGFß and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFß and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. CONCLUSIONS: Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade.

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.

Blockade of surface-bound TGF-ß on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment.

Regulatory T cells (Tregs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8+ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from Treg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor-ß (TGF-ß) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident Tregs in mice. Tregs isolated from B16 tumors alone were sufficient to suppress CD8+ T cell-mediated killing, which depended on surface-bound TGF-ß on the Tregs Immunosuppression of CD8+ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between Tregs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-ß-dependent manner and highlight potential ways to inhibit intratumoral Tregs therapeutically.

Timing of CSF-1/CSF-1R signaling blockade is critical to improving responses to CTLA-4 based immunotherapy.

UNLABELLED: Colony stimulating factor-1 (CSF-1) is produced by a variety of cancers and recruits myeloid cells that suppress antitumor immunity, including myeloid-derived suppressor cells (MDSCs.) Here, we show that both CSF-1 and its receptor (CSF-1R) are frequently expressed in tumors from cancer patients, and that this expression correlates with tumor-infiltration of MDSCs. Furthermore, we demonstrate that these tumor-infiltrating MDSCs are highly immunosuppressive but can be reprogrammed toward an antitumor phenotype in vitro upon CSF-1/CSF-1R signaling blockade. Supporting these findings, we show that inhibition of CSF-1/CSF-1R signaling using an anti-CSF-1R antibody can regulate both the number and the function of MDSCs in murine tumors in vivo. We further find that treatment with anti-CSF-1R antibody induces antitumor T-cell responses and tumor regression in multiple tumor models when combined with CTLA-4 blockade therapy. However, this occurs only when administered after or concurrent with CTLA-4 blockade, indicating that timing of each therapeutic intervention is critical for optimal antitumor responses. Importantly, MDSCs present within murine tumors after CTLA-4 blockade showed increased expression of CSF-1R and were capable of suppressing T cell proliferation, and CSF-1/CSF-1R expression in the human tumors was not reduced after treatment with CTLA-4 blockade immunotherapy. Taken together, our findings suggest that CSF-1R-expressing MDSCs can be targeted to modulate the tumor microenvironment and that timing of CSF-1/CSF-1R signaling blockade is critical to improving responses to checkpoint based immunotherapy. SIGNIFICANCE: Infiltration by immunosuppressive myeloid cells contributes to tumor immune escape and can render patients resistant or less responsive to therapeutic intervention with checkpoint blocking antibodies. Our data demonstrate that blocking CSF-1/CSF-1R signaling using a monoclonal antibody directed to CSF-1R can regulate both the number and function of tumor-infiltrating immunosuppressive myeloid cells. In addition, our findings suggest that reprogramming myeloid responses may be a key in effectively enhancing cancer immunotherapy, offering several new potential combination therapies for future clinical testing. More importantly for clinical trial design, the timing of these interventions is critical to achieving improved tumor protection.

Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy.

With the success of ipilimumab and promise of programmed death-1 pathway-targeted agents, the field of tumor immunotherapy is expanding rapidly. Newer targets for clinical development include select members of the tumor necrosis factor receptor (TNFR) family. Agonist antibodies to these co-stimulatory molecules target both T and B cells, modulating T-cell activation and enhancing immune responses. In vitro and in vivo preclinical data have provided the basis for continued development of 4-1BB, OX40, glucocorticoid-induced TNFR-related gene, herpes virus entry mediator, and CD27 as potential therapies for patients with cancer. In this review, we summarize the immune response to tumors, consider preclinical and early clinical data on select TNFR family members, discuss potential translational challenges and suggest possible combination therapies with the aim of inducing durable antitumor responses.

Biomarkers for immunostimulatory monoclonal antibodies in combination strategies for melanoma and other tumor types.

Modulation of the immune system by targeting coinhibitory and costimulatory receptors has become a promising new approach of immunotherapy for cancer. The recent approval of the CTLA-4-blocking antibody ipilimumab for the treatment of melanoma was a watershed event, opening up a new era in the field of immunotherapy. Ipilimumab was the first treatment to ever show enhanced overall survival (OS) for patients with stage IV melanoma. However, measuring response rates using standard Response Evaluation Criteria in Solid Tumors (RECIST) or modified World Health Organization criteria or progression-free survival does not accurately capture the potential for clinical benefit for ipilimumab-treated patients. As immunotherapy approaches are translated into more tumor types, it is important to study biomarkers, which may be more predictive of OS to identify the patients most likely to have clinical benefit. Ipilimumab is the first-in-class of a series of immunomodulating antibodies that are in clinical development. Anti-PD1 (nivolumab and MK-3475), anti-PD-L1 (BMS-936 559, RG7446, and MEDI4736), anti-CD137 (urelumab), anti-OX40, anti-GITR, and anti-CD40 monoclonal antibodies are just some of the agents that are being actively investigated in clinical trials, each having the potential for combination with the ipilimumab to enhance its effectiveness. Development of rational combinations of immunomodulatory antibodies with small-molecule pathway inhibitor therapies such as vemurafenib makes the discovery of predictive biomarkers even more important. Identifying reliable biomarkers is a necessary step in personalizing the treatment of each patient's cancer through a baseline assessment of tumor gene expression and/or immune profile to optimize therapy for the best chance of therapeutic success.

GITR pathway activation abrogates tumor immune suppression through loss of regulatory T cell lineage stability.

Ligation of GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor-related gene, or TNFRSF18) by agonist antibody has recently entered into early phase clinical trials for the treatment of advanced malignancies. Although the ability of GITR modulation to induce tumor regression is well-documented in preclinical studies, the underlying mechanisms of action, particularly its effects on CD4(+)foxp3(+) regulatory T cells (Treg), have not been fully elucidated. We have previously demonstrated that GITR ligation in vivo by agonist antibody DTA-1 causes a >50% reduction of intra-tumor Treg with down modulation of Foxp3 expression. Here we show that the loss of Foxp3 is tumor-dependent. Adoptively-transferred Foxp3(+)Treg from tumor-bearing animals lose Foxp3 expression in the host when treated with DTA-1, whereas Treg from naïve mice maintain Foxp3 expression. GITR ligation also alters the expression of various transcription factors and cytokines important for Treg function. Complete Foxp3 loss in intra-tumor Treg correlates with a dramatic decrease in Helios expression and is associated with the upregulation of transcription factors T-Bet and Eomes. Changes in Helios correspond with a reduction in IL-10 and an increase in IFNγ expression in DTA-1-treated Treg. Together, these data show that GITR agonist antibody alters Treg lineage stability inducing an inflammatory effector T cell phenotype. The resultant loss of lineage stability causes Treg to lose their intra-tumor immune suppressive function, making the tumor susceptible to killing by tumor-specific effector CD8(+) T cells.

Modulation of GITR for cancer immunotherapy.

Modulation of co-inhibitory and co-stimulatory receptors of the immune system has become a promising new approach for immunotherapy of cancer. With the recent FDA approval of CTLA-4 blockade serving as an important proof of principal, many new targets are now being translated into the clinic. Preclinical research has demonstrated that targeting glucocorticoid-induced tumor necrosis factor (TNF) receptor related gene (GITR), a member of TNF receptor superfamily, by agonist antibodies or natural ligand, can serve as an effective anti-tumor therapy. In this review, we will cover this research and the rationale that has led to initiation of two phase 1 clinical trials targeting GITR as a new immunotherapeutic approach for cancer.

Hiding the road signs that lead to tumor immunity.

Tumors exploit many strategies to evade T cell-mediated destruction. For example, tumors can prevent T cell infiltration by modifying gene expression in the endothelial cells and pericytes that form their vasculature. New work showing that the T cell-attracting chemokine CCL2 can be posttranslationally modified in the tumor microenvironment adds another mechanism to the already formidable arsenal of immunoevasion tactics used by solid tumors.

Detection of intra-tumor self antigen recognition during melanoma tumor progression in mice using advanced multimode confocal/two photon microscope.

Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response "functions" in vivo without therapeutic intervention. To better understand this question, we developed advanced multimodal reflectance confocal/two photon fluorescence intra-vital imaging techniques to use in combination with traditional ex vivo analysis of tumor specific T cells. By transferring small numbers of melanoma-specific CD8+ T cells (Pmel-1), in an attempt to mimic physiologic conditions, we found that B16 tumor growth alone was sufficient to induce naive Pmel-1 T cell proliferation and acquisition of effector phenotype. Tumor -primed Pmel-1 T cells, are capable of killing target cells in the periphery and secrete IFNγ, but are unable to mediate tumor regression. Within the tumor, Pmel-1 T cells have highly confined mobility, displaying long term interactions with tumor cells. In contrast, adoptively transferred non tumor-specific OT-I T cells show neither confined mobility, nor long term interaction with B16 tumor cells, suggesting that intra-tumor recognition of cognate self antigen by Pmel-1 T cells occurs during tumor growth. Together, these data indicate that lack of anti-tumor efficacy is not solely due to ignorance of self antigen in the tumor microenvironment but rather to active immunosuppressive influences preventing a protective immune response.

Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma.

Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine-glycine-aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvß3 integrin-expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting.

Modulation of CTLA-4 and GITR for cancer immunotherapy.

The rational manipulation of antigen-specific T cells to reignite a tumor-specific immune response in cancer patients is a challenge for cancer immunotherapy. Targeting coinhibitory and costimulatory T cell receptors with specific antibodies in cancer patients is an emerging approach to T cell manipulation, namely "immune modulation." Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor family receptor (GITR) are potential targets for immune modulation through anti-CTLA-4 blocking antibodies and anti-GITR agonistic antibodies, respectively. In this review, we first discuss preclinical findings key to the understanding of the mechanisms of action of these immunomodulatory antibodies and the preclinical evidence of antitumor activity which preceded translation into the clinic. We next describe the outcomes and immune related adverse effects associated with anti-CTLA-4 based clinical trials with particular emphasis on specific biomarkers used to elucidate the mechanisms of tumor immunity in patients. The experience with anti-CTLA-4 therapy and the durable clinical benefit observed provide proof of principle to effective antitumor immune modulation and the promise of future clinical immune modulatory antibodies.

Anti-GITR antibodies--potential clinical applications for tumor immunotherapy.

Since the development of the first vaccines, modern medicine has been consistently aiming to improve the efficacy of immune responses. Traditionally, adjuvants have been used as non-specific immune modulators to enhance recognition and activation against a desired antigen. By providing 'danger' signals to the immune system, adjuvants activate innate immunity, which enhances the development of protective and therapeutic adaptive immune responses. The newest class of immune modulators bypasses the innate response and targets cells of the adaptive response directly. Targeted immunomodulatory therapy is focused primarily on the activation of costimulatory receptors (eg, 4-1BB, OX40 and GITR [glucocorticoid-induced TNF receptor-related gene]) or the blockade of co-inhibitory receptors (eg, CTLA-4, PD-1 and PD-L1) on T-cells during activation and/or effector responses. With promising clinical results obtained to date, immunomodulatory therapy is becoming an integral part of immunotherapeutic approaches. The modulation of GITR is listed as one of the top 25 most promising research areas by the NCI, and has demonstrated potential in both antitumor and vaccine settings. This review discusses the role of GITR as a potential target for immunomodulatory therapy, as well as the research involved in understanding the mechanisms of anti-GITR therapy and current progress in translation into the clinic.