Multipeptide stimulated PBMCs generate TEM/TCM for adoptive cell therapy in multiple myeloma.

Multiple Myeloma (MM) patients suffer disease relapse due to the development of therapeutic resistance. Increasing evidence suggests that immunotherapeutic strategies can provide durable responses. Here we evaluate the possibility of adoptive cell transfer (ACT) by generating ex vivo T cells from peripheral blood mononuclear cells (PBMCs) isolated from MM patients by employing our previously devised protocols. We designed peptides from antigens (Ags) including cancer testis antigens (CTAs) that are over expressed in MM. We exposed PBMCs from different healthy donors (HDs) to single peptides. We observed reproducible Ag-specific cluster of differentiation 4+ (CD4+) and CD8+ T cell responses on exposure of PBMCs to different single peptide sequences. These peptide sequences were used to compile four different peptide cocktails. Naïve T cells from PBMCs from MM patients or HDs recognized the cognate Ag in all four peptide cocktails, leading to generation of multiclonal Ag-specific CD4+ and CD8+ effector and central memory T (TEM and TCM, respectively) cells which produced interferon-gamma (IFN-γ), granzyme B and perforin on secondary restimulation. Furthermore, this study demonstrated that immune cells from MM patients are capable of switching metabolic programs to induce effector and memory responses. Multiple peptides and cocktails were identified that induce IFN-γ+, T1-type, metabolically active T cells, thereby paving the way for feasibility testing of ACT in phase I clinical trials.

Synthesis and Immunological Evaluation of a Multicomponent Cancer Vaccine Candidate Containing a Long MUC1 Glycopeptide.

A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4+ T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.

Comparative analysis of condom lubricants on pre & post-coital vaginal swabs using AccuTOF-DART.

In this study we demonstrate the use of Direct Analysis in Real Time Mass Spectrometry (DART) as a powerful tool for detection of nonoxynol in vaginal fluid post contact with a condom, enabling rapid tracing and added evidences in sexual assault crimes. Vaginal fluid was sampled using cotton swabs and glass rods and measured directly with DART. Sample preparation using water, hexane, methanol, and dichloromethane extraction, was explored for comparison and optimization of signals. Nonoxynol was detected up to eight hours after sampling. Optimal sampling conditions and mass spectrometry parameters are reported and discussed.

Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment.

We have previously reported that direct injection of dendritic cells (DC) engineered to express the Type-1 transactivator Tbet (i.e., DC.Tbet) into murine tumors results in antitumor efficacy in association with the development of structures resembling tertiary lymphoid organs (TLO) in the tumor microenvironment (TME). These TLO contained robust infiltrates of B cells, DC, NK cells, and T cells in proximity to PNAd+ blood vessels; however, they were considered incomplete, since the recruited B cells failed to organize into classic germinal center-like structures. We now report that antitumor efficacy and TLO-inducing capacity of DC.Tbet-based i.t. therapy is operational in peripheral lymph node-deficient LTA-/- mice, and that it is highly dependent upon a direct Tbet target gene product, IL-36γ/IL-1F9. Intratumoral DC.Tbet fails to provide protection to tumor-bearing IL-36R-/- hosts, or to tumor-bearing wild-type recipient mice co-administered rmIL-1F5/IL-36RN, a natural IL-36R antagonist. Remarkably, the injection of tumors with DC engineered to secrete a bioactive form of mIL-36γ (DC.IL36γ) also initiated therapeutic TLO and slowed tumor progression in vivo. Furthermore, DC.IL36γ cells strongly upregulated their expression of Tbet, suggesting that Tbet and IL-36γ cooperate to reinforce each other's expression in DC, rendering them competent to promote TLO formation in an "immunologically normalized," therapeutic TME.

Chemotherapy can induce weight normalization of morbidly obese mice despite undiminished ingestion of high fat diet.

Morbidly obese patients who accomplish substantial weight loss often display a long-term decline in their resting metabolism, causing even relatively restrained caloric intake to trigger a relapse to the obese state. Paradoxically, we observed that morbidly obese mice receiving chemotherapy for cancer experienced spontaneous weight reduction despite unabated ingestion of their high fat diet (HFD). This response to chemotherapy could also be achieved in morbidly obese mice without cancer. Optimally dosed methotrexate (MTX) or cyclophosphamide (CY) enabled the mice to completely and safely normalize their body weight despite continued consumption of obesogenic quantities of HFD. Weight reduction was not attributable to decreased HFD intake, enhanced energy expenditure or malabsorption. MTX or CY dosing significantly depleted both adipose tissue and preadipocyte progenitors. Remarkably, however, despite continued high fat feeding, a compensatory increase in hepatocyte lipid storage was not observed, but rather the opposite. Gene microarray liver analyses demonstrated that HFD mice receiving MTX or CY experienced significantly inhibited lipogenesis and lipid storage, whereas Enho (energy homeostasis) gene expression was significantly upregulated. Further metabolic studies employing a human hepatocellular line revealed that MTX treatment preserved robust oxidative phosphorylation, but also promoted mitochondrial uncoupling with a surge in proton leak. This is the first report that certain optimally dosed chemotherapeutic agents can induce weight loss in morbidly obese mice without reduced dietary intake, apparently by depleting stores of adipocytes and their progenitors, curtailment of lipogenesis, and inconspicuous disposal of incoming dietary lipid via a steady state partial uncoupling of mitochondrial oxidative phosphorylation.

Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.

Effective adoptive immunotherapy has proved elusive for many types of human cancer, often due to difficulties achieving robust expansion of natural tumor-specific T-cells from peripheral blood. We hypothesized that antigen-driven T-cell expansion might best be triggered in vitro by acute activation of innate immunity to mimic a life-threatening infection. Unfractionated peripheral blood mononuclear cells (PBMC) were subjected to a two-step culture, first synchronizing their exposure to exogenous antigens with aggressive surrogate activation of innate immunity, followed by γ-chain cytokine-modulated T-cell hyperexpansion. Step 1 exposure to GM-CSF plus paired Toll-like receptor agonists (resiquimod and LPS), stimulated abundant IL-12 and IL-23 secretion, as well as upregulated co-stimulatory molecules and CD11c expression within the myeloid (CD33+) subpopulation. Added synthetic long peptides (>20aa) derived from widely expressed oncoproteins (MUC1, HER2/neu and CMVpp65), were reliably presented to CD4+ T-cells and cross-presented to CD8+ T-cells. Both presentation and cross-presentation demonstrated proteasomal and Sec61 dependence that could bypass the endoplasmic reticulum. Step 2 exposure to exogenous IL-7 or IL-7+IL-2 produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific T-cells with a predominant interferon-γ-producing T1-type, as well as the antigen-specific ability to lyse tumor targets. Other γ-chain cytokines and/or combinations were initially proliferogenic, but followed by a contractile phase not observed with IL-7 or IL-7+IL-2. Regulatory T-cells were minimally propagated under these culture conditions. This mechanistically rational culture sequence, effective even for unvaccinated donors, enables rapid preparation of T-cells recognizing tumor-associated antigens expressed by the majority of human cancers, including pancreatic cancers, breast cancers and glioblastomas.

Myeloid-Derived Suppressor Cell Subset Accumulation in Renal Cell Carcinoma Parenchyma Is Associated with Intratumoral Expression of IL1ß, IL8, CXCL5, and Mip-1α.

Purpose: Little is known about the association between myeloid-derived suppressor cell (MDSC) subsets and various chemokines in patients with renal cell carcinoma (RCC) or the factors that draw MDSC into tumor parenchyma.Experimental Design: We analyzed polymorphonuclear MDSC (PMN-MDSC), monocytic MDSC (M-MDSC), and immature MDSC (I-MDSC) from the parenchyma and peripheral blood of 48 patients with RCC, isolated at nephrectomy. We analyzed levels of IL1ß, IL8, CXCL5, Mip-1α, MCP-1, and Rantes. Furthermore, we performed experiments in a Renca murine model to assess therapeutic synergy between CXCR2 and anti-PD1 and to elucidate the impact of IL1ß blockade on MDSC.Results: Parenchymal PMN-MDSC have a positive correlation with IL1ß, IL8, CXCL5, and Mip-1α, and I-MDSC correlate with IL8 and CXCL5. Furthermore, peripheral PMN-MDSC correlate with tumor grade. Given that PMN-MDSC express CXCR2 and parenchymal PMN-MDSC correlated with IL8 and CXCL5, we assessed the response of CXCR2 blockade with or without anti-PD1. Combination therapy reduced tumor weight and enhanced CD4+ and CD8+ T-cell infiltration. In addition, anti-IL1ß decreased PMN-MDSC and M-MDSC in the periphery, PMN-MDSC in the tumor, and peripheral CXCL5 and KC. Anti-IL1ß also delayed tumor growth.Conclusions: Parenchymal PMN-MDSC have a positive correlation with IL1ß, IL8, CXCL5, and Mip-1α, suggesting they may attract PMN-MDSC into the tumor. Peripheral PMN-MDSC correlate with tumor grade, suggesting prognostic significance. Anti-CXCR2 and anti-PD1 synergized to reduce tumor weight and enhanced CD4+ and CD8+ T-cell infiltration in a Renca murine model, suggesting that CXCR2+ PMN-MDSC are important in reducing activity of anti-PD1 antibody. Finally, anti-IL1ß decreases MDSC and delayed tumor growth, suggesting a potential target for MDSC inhibition. Clin Cancer Res; 23(9); 2346-55. ©2016 AACR.

Sticking to (first) principles: quantum molecular dynamics and Bayesian probabilistic methods to simulate aquatic pollutant absorption spectra.

This work explores the relationship between theoretically predicted excitation energies and experimental molar absorption spectra as they pertain to environmental aquatic photochemistry. An overview of pertinent Quantum Chemical descriptions of sunlight-driven electronic transitions in organic pollutants is presented. Second, a combined molecular dynamics (MD), time-dependent density functional theory (TD-DFT) analysis of the ultraviolet to visible (UV-Vis) absorption spectra of six model organic compounds is presented alongside accurate experimental data. The functional relationship between the experimentally observed molar absorption spectrum and the discrete quantum transitions is examined. A rigorous comparison of the accuracy of the theoretical transition energies (ΔES0→Sn) and oscillator strength (fS0→Sn) is afforded by the probabilistic convolution and deconvolution procedure described. This method of deconvolution of experimental spectra using a Gaussian Mixture Model combined with Bayesian Information Criteria (BIC) to determine the mean (µ) and standard deviation (σ) as well as the number of observed singlet to singlet transition energy state distributions. This procedure allows a direct comparison of the one-electron (quantum) transitions that are the result of quantum chemical calculations and the ensemble of non-adiabatic quantum states that produce the macroscopic effect of a molar absorption spectrum. Poor agreement between the vertical excitation energies produced from TD-DFT calculations with five different functionals (CAM-B3LYP, PBE0, M06-2X, BP86, and LC-BLYP) suggest a failure of the theory to capture the low energy, environmentally important, electronic transitions in our model organic pollutants. However, the method of explicit-solvation of the organic solute using the quantum Effective Fragment Potential (EFP) in a density functional molecular dynamics trajectory simulation shows promise as a robust model of the hydrated organic pollutant. Furthermore, the described protocol can be extended using higher-level equilibration and vertical excitation methods to increase the numerical accuracy and describe multi-reference electronic transitions. Finally, a measure of the accuracy of theoretically derived absorption spectra is discussed as a tool to further develop our capacity to produce accurate a priori simulations of sunlight-driven photochemistry in natural waters.

Definitive activation of endogenous antitumor immunity by repetitive cycles of cyclophosphamide with interspersed Toll-like receptor agonists.

Many cancers both evoke and subvert endogenous anti-tumor immunity. However, immunosuppression can be therapeutically reversed in subsets of cancer patients by treatments such as checkpoint inhibitors or Toll-like receptor agonists (TLRa). Moreover, chemotherapy can leukodeplete immunosuppressive host elements, including myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). We hypothesized that chemotherapy-induced leukodepletion could be immunopotentiated by co-administering TLRa to emulate a life-threatening infection. Combining CpG (ODN 1826) or CpG+poly(I:C) with cyclophosphamide (CY) resulted in uniquely well-tolerated therapeutic synergy, permanently eradicating advanced mouse tumors including 4T1 (breast), Panc02 (pancreas) and CT26 (colorectal). Definitive treatment required endogenous CD8+ and CD4+ IFNγ-producing T-cells. Tumor-specific IFNγ-producing T-cells persisted during CY-induced leukopenia, whereas Tregs were progressively eliminated, especially intratumorally. Spleen-associated MDSCs were cyclically depleted by CY+TLRa treatment, with residual monocytic MDSCs requiring only continued exposure to CpG or CpG+IFNγ to effectively attack malignant cells while sparing non-transformed cells. Such tumor destruction occurred despite upregulated tumor expression of Programmed Death Ligand-1, but could be blocked by clodronate-loaded liposomes to deplete phagocytic cells or by nitric oxide synthase inhibitors. CY+TLRa also induced tumoricidal myeloid cells in naive mice, indicating that CY+TLRa's immunomodulatory impacts occurred in the complete absence of tumor-bearing, and that tumor-induced MDSCs were not an essential source of tumoricidal myeloid precursors. Repetitive CY+TLRa can therefore modulate endogenous immunity to eradicate advanced tumors without vaccinations or adoptive T-cell therapy. Human blood monocytes could be rendered similarly tumoricidal during in vitro activation with TLRa+IFNγ, underscoring the potential therapeutic relevance of these mouse tumor studies to cancer patients.

MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice.

It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.

Late-Stage Cancer Patients Remain Highly Responsive to Immune Activation by the Selective TLR8 Agonist Motolimod (VTX-2337).

PURPOSE: Immunotherapy as a treatment for cancer holds the promise of complete and durable tumor remission, yet the immunosuppressive environment created by many tumors, advanced patient age, and previous treatments with cytotoxic agents may limit the approach. The activity of motolimod (VTX-2337), a potent and selective Toll-like receptor 8 (TLR8) agonist, was therefore assessed in the context of advanced, late-stage cancer patients. EXPERIMENTAL DESIGN: The repertoire of mediators induced from human peripheral blood mononuclear cells in response to motolimod was characterized. Translational studies in cynomolgus monkeys elucidated the activity of motolimod on an intact immune system, identified biomarkers of TLR8 activation, and defined the relationship between the pharmacokinetic and pharmacodynamic (PK/PD) response. The PK/PD relationship for motolimod in cancer patients was assessed, compared with preclinical findings, and contrasted with activity in healthy volunteers. RESULTS: In late-stage cancer patients, plasma levels of multiple biomarkers, including IL6, G-CSF, MCP-1, and MIP1-ß, increased with increasing motolimod dose. The magnitude and breadth of the biomarker response closely aligned with the response seen in preclinical studies, demonstrating that advanced cancer patients remained responsive to TLR8 activation. In addition, the PK/PD response in cancer patients closely aligned with the activity of motolimod seen in healthy volunteers. CONCLUSIONS: Late-stage cancer patients are highly sensitive to TLR8 activation by motolimod. Tumor burden, advanced age, and prior treatment history with cytotoxic agents did not moderate or modify the response predicted by nonclinical studies and confirmed in healthy volunteers. Clin Cancer Res; 21(24); 5445-52. ©2015 AACR.

Linear synthesis and immunological properties of a fully synthetic vaccine candidate containing a sialylated MUC1 glycopeptide.

A strategy for the linear synthesis of a sialylated glycolipopeptide cancer vaccine candidate has been developed using a strategically designed sialyl-Tn building block and microwave-assisted solid-phase peptide synthesis. The glycolipopeptide elicited potent humoral and cellular immune responses. T-cells primed by such a vaccine candidate could be restimulated by tumor-associated MUC1.

Inhibition of adaptive immunity by IL9 can be disrupted to achieve rapid T-cell sensitization and rejection of progressive tumor challenges.

The tolerogenic cytokine IL9 promotes T regulatory cell function and allergic airway inflammation, but it has not been extensively studied in cancer. In this report, we used IL9-deficient mice to investigate the effects of IL9 in multiple models of breast and colon cancer development. Eliminating endogenous IL9 enabled sensitization of host T cells to tumors, leading to their early rejection without the requirement of vaccines or immunomodulatory therapies. Notably, IL9-deficient mice acquired immunologic memory, which actively protected from residual disease and tumor rechallenge, an effect linked to activation of CD8(+) T cells. Depletion of either CD8(+) or CD4(+) T cells abolished the benefits of IL9 loss to tumor control. Adoptive transfer experiments showed that T cells from tumor-rejecting IL9-deficient mice retained their effector competency in wild-type animals. Moreover, neutralizing IL9 antibody phenocopied the effects of IL9 gene deletion by slowing tumor progression in wild-type animals. Our results show the ability of IL9 to function as an inhibitor of adaptive immunity that prevents the formation of immunologic memory to a growing tumor, highlighting the potential for IL9 neutralization as a unique tool for cancer immunotherapy.

Immune and anticancer responses elicited by fully synthetic aberrantly glycosylated MUC1 tripartite vaccines modified by a TLR2 or TLR9 agonist.

The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3 CysSK4 ) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3 CysSK4 -containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3 CysSK4 , which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1.

A phase I dose-finding study of the novel Toll-like receptor 8 agonist VTX-2337 in adult subjects with advanced solid tumors or lymphoma.

PURPOSE: This phase I, open-label, uncontrolled, ascending-dose study explored the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacology of the TLR8 agonist VTX-2337 in subjects with advanced solid tumors or lymphoma. EXPERIMENTAL DESIGN: VTX-2337 doses (0.1-3.9 mg/m(2)) were administered subcutaneously on days 1, 8, and 15 of each 28-day cycle. Safety/tolerability assessments included adverse events (AE); physical, ophthalmologic, and laboratory evaluations; and electrocardiograms. Dose-limiting toxicities (DLT) were evaluated during the first cycle. Pharmacokinetics were evaluated after the first dose. Plasma samples were quantitatively assessed for chemokines, cytokines, and other inflammatory mediators. Antitumor activity was assessed. RESULTS: Thirty-three subjects were enrolled in 8 cohorts and received an average of 2 treatment cycles (range, 1-8 cycles). Most AEs were grades 1 to 2; the most common drug-related AEs were injection site reactions, chills, pyrexia, and influenza-like illness. One DLT was reported: grade 3 hypotension (3.9 mg/m(2)). The MTD was considered the highest dose administered. Peak drug plasma levels and total systemic exposure were generally dose proportional. At doses ≥0.4 mg/m(2), increases above baseline levels were observed for plasma levels of G-CSF, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and TNFα. Eight subjects (24.2%) had a best response of stable disease (median duration, 54.5 days). CONCLUSIONS: VTX-2337 is clinically well tolerated and biologically active with a predictable pharmacokinetic profile. Suitable doses for testing in combination studies were identified. Phase II placebo-controlled studies of VTX-2337 in combination with doxorubicin in ovarian cancer, and in combination with platinum chemotherapy, 5 FU, and cetuximab in head and neck cancer have been initiated (NCT #01666444 and NCT#01836029).

Adoptive transfer of MART-1 T-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma.

PURPOSE: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy. EXPERIMENTAL DESIGN: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation. RESULTS: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells. CONCLUSION: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.

Combination of sunitinib with anti-tumor vaccination inhibits T cell priming and requires careful scheduling to achieve productive immunotherapy.

Sunitinib, a protein tyrosine kinase inhibitor is the frontline therapy for renal and gastrointestinal cancers. We hypothesized that by virtue of its well documented tumor apoptosis and immune adjuvant properties, combination of Sunitinib with anti-tumor immunotherapeutics will provide synergistic inhibition of tumor growth. Our study was designed to evaluate the impact of Sunitinib on immunotherapy mediated anti-tumor immune responses and evaluate its efficacy as a combinatorial therapy with tumor targeted immunotherapeutic vaccination. Mice immunized with recombinant α-lactalbumin, a lactation protein expressed on majority of breast tumors were treated with 1 mg of Sunitinib for seven consecutive days beginning (1) concurrently, on the day of α-lactalbumin immunization or (2) sequentially, on day 9 after immunization. Ten-day lymph nodes or 21 day spleens were tested by ELISPOT assays and flow cytometry to evaluate responsiveness to α-lactalbumin immunization in presence of Sunitinib and distribution of cells involved in T cell antigen priming and proliferation in different lymphoid compartments. In addition, therapeutic efficacy of the α-lactalbumin/ Sunitinib combination was evaluated by monitoring tumor growth in the 4T1 transplanted tumor model. Our studies reveal that concurrent administration of Sunitinib with active vaccination against a targeted tumor antigen inhibits priming to the immunogen due to a drastic decrease in CD11b+CD11c+ antigen presenting cells, leading to failure of vaccination. However, sequential delivery of Sunitinib timed to avoid the priming phase of vaccination results in the desired vaccination mediated boost in immune responses.

Modification of the tumor microenvironment as a novel target of renal cell carcinoma therapeutics.

To move forward with immunotherapy, it is important to understand how the tumor microenvironment generates systemic immunosuppression in patients with renal cell carcinoma (RCC) as well as in patients with other types of solid tumors. Even though antigen discovery in RCC has lagged behind melanoma, recent clinical trials have finally authenticated that RCC is susceptible to vaccine-based therapy. Furthermore, judicious coadministration of cytokines and chemotherapy can potentiate therapeutic responses to vaccine in RCC and prolong survival, as has already proved possible for melanoma. Although high-dose interleukin 2 immunotherapy has been superseded as first-line therapy for RCC by promiscuous receptor tyrosine kinase inhibitors (rTKIs) such as sunitinib, sunitinib itself is a potent immunoadjunct in animal tumor models. A reasonable therapeutic goal is to unite antiangiogenic strategies with immunotherapy as first-line therapy for RCC. This strategy is equally appropriate for testing in all solid tumors in which the microenvironment generates immunosuppression. A common element of RCC and pancreatic, colon, breast, and other solid tumors is large numbers of circulating myeloid-derived suppressor cells (MDSCs), and because MDSCs elicit regulatory T cells rather than vice versa, gaining control over MDSCs is an important initial step in any immunotherapy. Although rTKIs like sunitinib have a remarkable capacity to deplete MDSCs and restore normal T-cell function in peripheral body compartments such as the bloodstream and the spleen, such rTKIs are effective only against MDSCs, which are engaged in phospho-STAT3-dependent programming (pSTAT3+). Unfortunately, rTKI-resistant pSTAT3- MDSCs are especially apt to arise within the tumor microenvironment itself, necessitating strategies that do not rely exclusively on STAT3 disruption. The most utilitarian strategy to gain control of both pSTAT3+ and pSTAT3- MDSCs may be to exploit the natural differentiation pathway, which permits MDSCs to mature into tumoricidal macrophages (TM1) via such stimuli as Toll-like receptor agonists, interferon γ, and CD40 ligation. Overall, this review highlights the mechanisms of immune suppression used by the different regulatory cell types operative in RCC as well as other tumors. It also describes the different therapeutic strategies to overcome the suppressive nature of the tumor microenvironment.