Eradication Of Established Tumors Research Articles

In vivo targeting of a tumor-antigen encoded DNA vaccine to dendritic cells in combination with tumor-selective chemotherapy eradicates established mouse melanoma.

Despite remarkable progress during the past decade, eradication of established tumors by targeted cancer therapy and cancer immunotherapy remains an uphill task. Herein, we report on a combination approach for eradicating established mouse melanoma. Our approach employs the use of tumor selective chemotherapy in combination with in vivo dendritic cell (DC) targeted DNA vaccination. Liposomes of a newly synthesized lipopeptide containing a previously reported tumor-targeting CGKRK-ligand covalently grafted in its polar head-group region were used for tumor selective delivery of cancer therapeutics. Liposomally co-loaded STAT3siRNA and WP1066 (a commercially available inhibitor of the JAK2/STAT3 pathway) were used as cancer therapeutics. In vivo targeting of a melanoma antigen (MART-1) encoded DNA vaccine (p-CMV-MART1) to dendritic cells was accomplished by complexing it with a previously reported mannose-receptor selective in vivo DC-targeting liposome. Liposomes of the CGKRK-lipopeptide containing encapsulated FITC-labeled siRNA, upon intravenous administration in B16F10 melanoma bearing mice, showed remarkably higher accumulation in tumors 24 h post i.v. treatment, compared to their degree of accumulation in other body tissues including the lungs, liver, kidneys, spleen and heart. Importantly, the findings in tumor growth inhibition studies revealed that only in vivo DC-targeted genetic immunization or only tumor-selective chemotherapy using the presently described systems failed to eradicate the established mouse melanoma. The presently described combination approach is expected to find future applications in combating various malignancies (with well-defined surface antigens).

Alphaviruses in Cancer Therapy.

Alphaviruses have been engineered as expression vectors for different strategies of cancer therapy including immunotherapy and cancer vaccine development. Administration of recombinant virus particles, RNA replicons and plasmid DNA-based replicons provide great flexibility for alphavirus applications. Immunization and delivery studies have demonstrated therapeutic efficacy in the form of reduced tumor growth, tumor regression and eradication of established tumors in different animal models for cancers such as brain, breast, colon, cervical, lung, ovarian, pancreas, prostate cancers, and melanoma. Furthermore, vaccinated animals have showed protection against challenges with tumor cells. A limited number of clinical trials in the area of brain, breast, cervical, colon prostate cancers and melanoma vaccines has been conducted. Particularly, immunization of cervical cancer patients elicited immune responses and therapeutic activity in all patients included in a phase I clinical trial. Moreover, stable disease and partial responses were observed in breast cancer patients and prolonged survival was achieved in colon cancer patients.

Abstract B178: Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy

Abstract Background: The first cancer immunotherapy was developed in the 1890s by Dr. William Coley, who observed tumor regressions after injecting cancer patients with a heat-killed mixture of Gram-positive and negative pathogenic bacteria. Dr. Coley determined that the Gram-negative bacteria were the principal contributor to antitumor activity and that the product would likely be most effective when administered intravenously (i.v.), but was too toxic in this setting, limiting use to intramuscular and intratumoral administration. Coley’s toxins, as it was known, was credited with curing hundreds of late-stage cancer patients over 70 years. However, lack of knowledge regarding mechanism of action made it difficult to optimize or standardize, producing high variability. The use of multiple, local administration routes also likely contributed to variability in response, leading the FDA to refuse to grandfather in the product in 1963. We now know the mechanism of action of Coley’s toxins and the source of the i.v. toxicity. Gram-negative bacteria contain immune system danger signals, including multiple TLR agonists (activating TLRs 2, 4, 5 and 9), which directly and indirectly, via induction of cytokine and chemokine secretion, participate in the activation of most of the cellular mediators of innate and adaptive immune responses. Lipopolysaccharide (LPS), which activates TLR4, has been identified as a major contributor to both the antitumor activity and i.v. toxicity of Gram-negative bacteria. Decoy’s hypothesis is that significant reduction without complete elimination of LPS activity, in conjunction with killing and stabilization of nonpathogenic, Gram-negative bacteria, may produce a multiple TLR product that can safely and effectively induce antitumor immune responses via i.v. administration. Methods and Results: Nonpathogenic, Gram-negative E. coli were treated with polymyxin B and glutaraldehyde under conditions to kill and stabilize the cells, producing >90% reduction in LPS endotoxin activity and pyrogenicity. Endotoxin activity and pyrogenicity were quantified using Limulus amebocyte lysate (LAL) and in vivo rabbit assays. Bacterial integrity was assessed by electron and light microscopy. Antitumor activity was determined using standard syngeneic and xenograft tumor models. Decoy-treated bacteria exhibited a 3-fold reduction in acute in vivo toxicity relative to untreated bacteria. Surprisingly, induction of antitumor cytokine secretion by murine and human peripheral blood mononuclear cells (PBMCs) was not compromised, relative to untreated bacteria. Treatment with Decoy bacteria (i.v.) produced significant single-agent antitumor activity against orthotopic murine colorectal carcinoma and metastatic murine pancreatic carcinoma. Synergistic combination activity, including eradication of established tumors, with a therapeutic index of up to 10-fold, was observed in combination with IL-2 or low-dose cyclophosphamide (LDC) in murine colorectal carcinoma models, with LDC in a subcutaneous (s.c.), murine non-Hodgkin’s lymphoma (NHL) model and with LDC plus rituximab in a s.c., human NHL model. Synergistic antitumor activity was also observed in combination with a low-dose, nonsteroidal anti-inflammatory drug (NSAID) in a metastatic, murine pancreatic carcinoma model. In addition, tumor eradications were observed in combination with NSAID and were enhanced by addition of anti-PD-1 therapy in a s.c., murine hepatocellular carcinoma model. Optimal (80-100%) tumor eradication was shown to be mediated by natural killer (NK), CD4+ and CD8+ T-cells. Immunologic memory (80-100% and partial), determined by rejection of subsequent tumor challenge, was demonstrated in both immune competent and innate only settings, respectively. Conclusion: We have developed an i.v. administered multiple TLR agonist, based on Coley’s toxins, that can be combined with a wide variety of existing agents to induce curative, antitumor immune responses. Citation Format: Michael J. Newman. Development and preclinical efficacy characterization of a systemically administered multiple Toll-like receptor (TLR) agonist for antitumor immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B178.

Abstract 2787: Generation of first-in-class anti-CD25 antibodies depleting Treg without interfering with IL2 signalling for cancer therapies

Abstract Regulatory T cells (Treg) are key players of the suppressive tumour microenvironment (TME). Their presence is correlated with a bad prognosis in multiple cancers, while a greater ratio of effector T cells (Teff) to Treg is associated with improved outcome. Studies demonstrating high expression of CD25 on Tregs but not Teff in human tumors have underscored its relevance as a target for Treg depletion. However, clinical trials conducted to target Treg in cancer patients with existing anti-CD25 (aCD25) antibodies have shown contradicting results, and no aCD25 antibody is in clinical development for this application. Importantly, aCD25 antibodies tested to date block IL2 signalling via CD25. We demonstrated (Solomon et al. AACR2018) that such blocking drastically reduces the therapeutic activity of aCD25 antibodies and that a single administration of a depleting antibody targeting CD25 but not blocking the IL2 signalling effectively depletes Treg, increases Teff activity and promotes eradication of established tumors in several mouse models of cancer. With this rationale in mind, we have generated a panel of fully human aCD25 IgG1 antibodies, which we characterized for their binding to human and cynomolgus monkey CD25. The antibodies were then screened for their impact on IL2 binding, using a sandwich binding assay on Octet, and on IL2 signalling, using a STAT5 phosphorylation assay. Antibodies were also tested for their ability to deplete CD25 positive cell lines and in vitro-derived human Treg in ADCC and ADCP assays. Finally, the impact of selected antibodies on Treg within the TME and on Teff responses was evaluated in human samples. Among the antibodies binding to human and cynomolgus CD25, we have selected a panel interfering with neither IL2 binding to CD25 nor IL2 signalling. Interestingly, epitope binning assays demonstrated that none of the selected antibodies bind to epitopes overlapping with those of the existing clinical antibodies, Daclizumab and Basiliximab. Among the non-IL2 blocking antibodies, those showing the maximum target cell lysis via ADCC and highest phagocytosis via ADCP represent potential lead clinical candidates. Most interestingly, contrary to the existing clinical aCD25 antibodies, which block IL2 signalling, our candidates do not inhibit Teff proliferation and function, confirming the specificity of action toward Treg and the importance of preserving IL2 signalling. Finally, we have shown that our clinical candidates efficiently deplete Treg in the TME. We present the first anti-human CD25 antibodies selected for their capacity to deplete human Treg while preserving IL2 signalling and activity of Teff. These antibodies provide a novel therapeutic approach to alleviate immune suppression in the TME, which would provide an ideal combination partner for existing standard of care and IO treatments, and could potentially be used as a monotherapy. Citation Format: Josephine C. Salimu, Mark Brown, Pascal Merchiers, Beatriz Goyenechea, Kevin Moulder, Robert Dejonge, Aghiles Boughetane, Isabelle Solomon, Frederick Arce Vargas, Karl S. Peggs, Anne Goubier, Sergio A. Quezada. Generation of first-in-class anti-CD25 antibodies depleting Treg without interfering with IL2 signalling for cancer therapies [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 2787.

Eradication of Established Tumors by Chemically Self-Assembled Nanoring Labeled T Cells.

Our laboratory has developed chemically self-assembled nanorings (CSANs) as prosthetic antigen receptors (PARs) for the nongenetic modification of T cell surfaces. PARs have been successfully employed in vitro to activate T cells for the selective killing of leukemia cells. However, PAR efficacy has yet to be evaluated in vivo or against solid tumors. Therefore, we developed bispecific PARs that selectively target the human CD3 receptor and human epithelial cell adhesion molecule (EpCAM), which is overexpressed on multiple carcinomas and cancer stem cells. The αEpCAM/αCD3 PARs were found to stably bind T cells for >4 days, and treating EpCAM+ MCF-7 breast cancer cells with αEpCAM/αCD3 PAR-functionalized T cells resulted in the induction of IL-2, IFN-γ, and MCF-7 cytotoxicity. Furthermore, an orthotopic breast cancer model validated the ability of αEpCAM/αCD3 PAR therapy to direct T cell lytic activity toward EpCAM+ breast cancer cells in vivo, leading to tumor eradication. In vivo biodistribution studies demonstrated that PAR-T cells were formed in vivo and persist for over 48 h with rapid accumulation in tumor tissue. Following PAR treatment, the production of IL-2, IFN-γ, IL-6, and TNF-α could be significantly reduced by an infusion of clinically relevant concentrations of the FDA-approved antibiotic, trimethoprim, signaling pharmacologic PAR deactivation. Importantly, CSANs did not induce naïve T cell activation and thus exhibit a limited potential to induce naïve T cell anergy. In addition, murine immunogenicity studies demonstrated that CSANs do not induce a significant antibody response nor do they activate splenic cells. Collectively, our results demonstrate that bispecific CSANs are able to nongenetically generate reversibly modified T cells that are capable of eradicating targeted solid tumors.

Abstract OT3-06-04: A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC)

Abstract Primary Objective: To determine pathological complete response (pCR) rate in patients with primary TNIBC treated with PaCT in comparison with CT, followed by AC. To explore if the pCR rate correlates with reduced nodal expression status; and with arginine methylation status of epidermal growth factor receptor (EGFR). We will identify molecular biomarkers predictive of the pCR rate by analysis of multiplexed immunohistochemical (IHC) staining, identify molecular biomarkers predictive of the pCR rate by genomic and proteomic analysis, and determine whether the inhibition of the EGFR pathway down regulates the COX-2 pathway and mesenchymal marker. Background: EGFR is overexpressed in triple negative breast cancer (TNBC) and inflammatory breast cancer (IBC). Therefore, EGFR targeted therapy may have a promising role in TNBC and IBC. A study showed that EGFR-targeted therapy may enhance the initial chemosensitivity of TNBC cells. Panitumumab blocks epidermal growth factor ligands and transforming growth factor EGFá (TGFá) binding to EGFR, inhibits tumor growth, and elicits both tumor regression and eradication of established tumors in murine xenograft tumor models. Panitumumab, a fully humanized anti-EGFR antibody, has been shown to be active in a breast cancer preclinical model using human breast cancer cell line MDA-MB-468, which has been shown to overexpress EGFR by both IHC and fluorescence in situ hybridization (FISH). Furthermore, EGFR tyrosine kinase inhibitors such as erlotinib have antitumor activity against human IBC cell lines. Thus, EGFR targeted therapy may have a promising role in TNBC and IBC. Study Design: In this open label randomized phase II trial, up to 72 patients with primary IBC, have no HER2 overexpression, and have <10% expression of ER and PgR, who also meet other criteria will be randomized to PaCT arm - receiving panitumumab single agent in window study and 4 cycles PaCT, or CT arm - receiving 4 cycles of CT. All patients will receive 4 cycles of AC before surgery. Statistical Considerations: A sample size of 36 patients per arm will achieve 84% power to detect a difference of 0.24 in pCR rate between 0.2 in the CT arm and 0.44 in the PaCT arm with a type I error rate of 10% using one-sided Z test. Based on historical data, we expect that the pCR rate of a PaCT regimen to achieve 24% additional efficacy compared with the CT regimen. Status of the study: Activation date: Oct. 2016. So far 6 patients have been enrolled. Enrollment continues. Sponsor: Amgen. State of Texas appropriation for rare and aggressive breast cancer research. NIH grant 1R01CA205043-01A1 Citation Format: Willey JS, Parker CA, Lim B, Valero V, Le-Petross HT, Krishnamurthy S, Woodward WA, Lucci A, Wood AL, Sun H, Babiera GV, Song J, Shen Y, Valero V, Wang X, Ueno NT. A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-06-04.

Abstract LB-491: PD-1 expression level on T cell subsets is a crucial factor forming a novel mechanism for designing immune therapeutic approaches using PD-1 binding ligand

Abstract A significant barrier to tumor regression in the setting of a successful immune response is tumor-mediated immunosuppression. Programmed death receptor-1 (PD-1) is an important signaling molecule and in the context of the tumor microenvironment may inhibit T cell immunity when bound to its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), by inducing T cell apoptosis and anergy. Recently we demonstrated that combination of anti-PD-1 antibody with vaccine and low-dose of cyclophosphamide (CPM), that reduces the number and function of Treg cells, significantly enhances antigen-specific immune responses, decreases tumor burden and increases survival of treated mice, suggesting that targeting multiple immune checkpoint inhibitors is a promising strategy for tumor immunotherapy. Here we tested the anti-tumor immune effect of B7-DC-Ig, a recombinant protein that binds and target PD-1. It is composed of the extracellular domain of B7-DC, fused to the Fc portion of murine IgG2a. We demonstrated that B7-DC-Ig can enhance the therapeutic efficacy of a peptide based cancer vaccine when combined with CPM. We showed that this combination significantly enhances antigen-specific immune responses and leads to complete eradication of established tumors in 60% of mice and that this effect is CD8 dependent. Importantly, we found that unlike anti-PD-1 antibody, B7-DC-Ig does not block the suppressive PD-1/PD-L1 interaction as its primary mechanism, and identified a novel mechanism by which B7-DC-Ig exerts its therapeutic effect. We demonstrate, for the first time, that there are significant differences between levels and timing of surface PD-1 expression on different T cell subsets. We found that these differences play a critical role in the anti-tumor effects exhibited by B7-DC-Ig through inhibiting proliferation of dysfunctional PD-1high CD4 T cells, for both Treg and FoxP3−T cells, leading to a significant decrease in levels of tumor infiltration by dysfunctional T cells. In addition, we showed that treatment with B7-DC-Ig also leads to a decrease in PD-1highCD8 T cells, tipping the balance towards non-exhausted, functional PD-1lowCD8 T cells. Aside from presenting a promising anti-tumor immunotherapeutic approach, we demonstrated that the level of PD-1 expression on different T cells subsets determines the “effective target zone” of B7-DC-Ig. We believe that this represents an important parameter that needs to be considered to favorably alter the tumor microenvironment when designing PD-1 targeting immune therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-491. doi:1538-7445.AM2012-LB-491

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

Selective expansion of merocytic dendritic cells and CD8DCs confers anti-tumour effect of Fms-like tyrosine kinase 3-ligand treatmentin vivo

Vaccination with autologous cancer cells aims to enhance adaptive immune responses to tumour-associated antigens. The incorporation of Fms-like tyrosine kinase 3-ligand (FLT3L) treatment to the vaccination scheme has been shown previously to increase the immunogenicity of cancer vaccines, thereby enhancing their therapeutic potential. While evidence has been provided that FLT3L confers its effect through the increase of absolute dendritic cell (DC) numbers, it is currently unknown which DC populations are responsive to FLT3L and which effect FLT3L treatment has on DC functions. Here we show that the beneficial effects of FLT3L treatment resulted predominantly from a marked increase of two specific DC populations, the CD8 DCs and the recently identified merocytic DC (mcDC). These two DC populations (cross)-present cell-associated antigens to T cells in a natural killer (NK)-independent fashion. FLT3L treatment augmented the absolute numbers of these DCs, but did not change their activation status nor their capacity to prime antigen-specific T cells. While both DC populations effectively primed CD8(+) T cell responses to cell-associated antigens, only mcDC were capable to prime CD4(+) T cells to cell-associated antigens. Consequentially, the transfer of tumour vaccine-pulsed mcDC, but not of CD8 DCs, protected mice from subsequent tumour challenge in a vaccination model and resulted in eradication of established tumours in a therapeutic approach. These results show that the beneficial effect of FLT3L is associated with the induction of mcDC and suggests that selective targeting to mcDC or instilling mcDC 'characteristics' into conventional DC populations could significantly enhance the efficacy of tumour vaccines.

CT-011, Anti PD-1 Antibody, Enhances Ex-Vivo T Cell Responses To Autologous Dendritic/Myeloma Fusion Vaccine Developed For The Treatment Of Multiple Myeloma

We have developed a cancer vaccine in which autologous myeloma cells are fused with dendritic cells (DCs) resulting in the presentation of tumor antigens in the context of DC mediated costimulation. In animal models, vaccination with fusion cells results in eradication of established tumor, and in clinical trials, both immunologic and clinical responses have been observed. However, response to vaccination may be muted by inhibitory signals such as the PD1/PDL1 pathway which blunt activated T cell responses.

CT-011, Anti-PD-1 Antibody, Enhances Ex-Vivo T Cell Responses to Autologous Dendritic/Myeloma Fusion Vaccine Developed for the Treatment of Multiple Myeloma.

Abstract 781We have developed a promising cancer vaccine in which autologous tumor cells are fused with dendritic cells (DCs) resulting in the presentation of tumor antigens in the context of DC mediated costimulation. In animal models, vaccination with fusion cells results in eradication of established tumor, and in clinical trials, both immunologic and clinical responses have been observed. However, response to vaccination may be muted by inhibitory pathways that blunt activated T cell responses. The PD-1/PDL-1 pathway is an important element contributing to tumor mediated immune suppression. In infectious disease models, upregulation of T cell expression of PD-1 is associated with an exhausted phenotype facilitating the development of chronic viral infection. In contrast, PD-1 blockade results in the restoration of functionally active T cells and clearance of infection. The PD-1/PDL-1 pathway is also being evaluated a as a central mechanism by which tumors escape host immunity. CT-011, is a humanized anti PD-1 antibody that is currently evaluated in Phase II studies for the treatment of hematological malignancies and solid tumors. In this study, we evaluated expression of PD-1 on T cells derived from patients with advanced hematologic malignancies, and PDL-1 expression on primary myeloma cells, ex-vivo generated dendritic cells, and DC/tumor fusion cells. We evaluated the effect of PD-1 blockade with CT-011 on T cell response to DC/tumor fusion cell stimulation in vitro. Tumor cells were obtained from bone marrow aspirates of patients with multiple myeloma. Nonadherent peripheral blood mononuclear cells obtained from patients with multiple myeloma and normal volunteers were cultured in RPMI supplemented with 10U/ml IL-2, and expression of PD-1 on CD4+ T cells was assessed by flow cytometric analysis . DCs were generated from adherent mononuclear cells cultured with rhIL-4, GM-CSF and TNFα and fused with tumor cells by coculture in 50% solution of polyethylene glycol. T cells were stimulated by DC/tumor fusions in the presence or absence of 5ug/ml CT-011. We demonstrate that PD-1 expression is markedly upregulated on T cells in patients with advanced multiple myeloma. As compared to a control population of normal volunteers in which mean levels of PD-1 expression was 6% (n=7), mean expression in patients with multiple myeloma was 20% (n=9). These findings suggest that upregulation of PD-1 expression may play a central role in tumor mediated immune suppression. Mean expression of PDL-1 was 91% on dendritic cells generated from adherent peripheral blood mononuclear cells obtained from normal volunteers (n=6), and 66% on patient derived myeloma cells (n=3). In addition, PDL-1 expression is found in greater than 90% of DC/tumor fusions (n=2), which potentially provides an inhibitory signal dampening fusion mediated immunologic response. We examined the effect of PD-1 blockade on T cell response to DC/tumor fusions ex vivo with different anti PD-1 antibodies including CT-011. DC/tumor fusions were co-cultured with autologous T cells alone or with antibodies against PD-1 Enhanced fusion mediated stimulation of T cells was noted particularly with CT-011, resulting in a greater than 5 fold increase in T cell proliferation. Interferon gamma secretion by CD4+ T cells in response to stimulation by DC/myeloma fusion cells was increased from 4% to 11% in the presence of CT-011. In addition, IL-10 secretion by CD4+ cells following DC/myeloma fusion stimulation decreased from 6.5% to 3.5% following PD-1 blocakde . In summary, we have demonstrated that PD-1 expression is increased in T cells of patients with hematologic malignancy, and CT-011, a PD-1 blocking antibody, enhances activated T cell responses following stimulation with a DC/tumor fusion vaccine. A clinical trial in which patients with multiple myeloma are treated with DC/myeloma fusions in conjunction with CT-011 following autologous transplantation is planned. Disclosures:Schickler:CureTech, Ltd.: Employment. Rotem-Yehudar:CureTech, Ltd.: Employment.

In vivo activity of rituximab-CpG oligodeoxynucleotide conjugate against rituximab-resistant human CD20+ B-cell lymphoma

8529 Background: The anti-CD20 monoclonal antibody rituximab (R) is a mainstay in the treatment of B cell non-Hodgkin's lymphoma (NHL), exerting anti-tumor effects via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG) are potent activators of ADCC and T cell immunity, and have been studied for anti-NHL effects when administered by systemic or intratumoral routes. We sought to optimize the delivery of CpG to sites of NHL to improve R efficacy. Methods: We utilized an aggressive syngeneic human CD20-expressing murine B cell lymphoma (38C13-huCD20) to study the in vivo augmentation of R efficacy by CpG. Established tumors in immunocompetent mice were treated with R plus CpG given systemically, intratumorally, or chemically linked to antibody using maleimide-sulfhydryl chemistry (D. Betting et al, J. Immunol. 2008; 181: 4131). Results: 5- to 7-day established tumors were completely resistant to single agent R. Combining intratumoral, but not systemic administration of CpG with R resulted in tumor eradication from up to 42% of mice (p < 0.0003 vs. R alone, CpG alone, R + systemic CpG). Mechanistic studies indicated that both natural killer cells and complement participated in the cure of tumors by intratumoral CpG + R, by increasing tumor cell sensitivity to complement and ADCC lysis, and by augmenting the cytotoxicity of ADCC effectors. To overcome the need for repeated direct intratumoral injections and allow precise targeting of CpG to tumor cells, we chemically linked CpG to R using a cleavable linker. A single i.v. injection of this R-CpG conjugate achieved eradication of established tumors from 100% of mice. In contrast, equivalent doses of unlinked i.v. R + CpG, CpG alone, or R alone cured only 8% of mice. Thus, combining CpG with R was most effective using direct conjugation to the antibody. Conclusions: In conclusion, enhancement of R efficacy required sustained intratumoral delivery of CpG to maximize anti-tumor responses. R-CpG conjugate efficiently eradicated an established B cell lymphoma that is fully resistant to single-agent R. Clinical testing of anti-CD20-CpG conjugates against B cell NHL is thus warranted. [Table: see text]

A novel form of 4-1BB ligand has better efficacy than agonistic 4-1BB antibody as well as TLR agonists in a peptide based cancer vaccine (41.5)

Abstract Toll like receptor (TLR) agonists, MPL and CpG-ODN, have been the subject of intense preclinical and clinical investigations as most promising vaccine adjuvants. Given the paramount importance of costimulation in the modulation of immune responses, we hypothesized that agonistic costimulatory ligands may serve effective alternatives to TLR agonists for vaccine development. Unlike TLR agonists, costimulatory ligands primarily function as cell membrane-bound proteins and lack activity in soluble form. We, therefore, used core streptavidin as a chaperon to generate costimulatory ligands that function in soluble forms. We compared the activity of a novel form of 4-1BBL composed of the extracellular domain of murine 4-1BBL fused to the C-terminus of core streptavidin (SA-4-1BBL) with an agonistic Abs to 4-1BB and TLR agonists as components of peptide based vaccines in a cervical cancer mouse model. Vaccination with SA-4-1BBL resulted in better efficacy than agonistic Ab as well as MPL/LPS/CpG in the eradication of established tumors. Therapeutic efficacy of SA-4-1BBL was associated with enhanced tumor specific primary and memory responses and reversal of T cell anergy. Importantly, therapeutic efficacy was achieved in the absence of sever toxicity associated with the agonistic Ab. Taken together, these data demonstrate the feasibility of using SA-4-1BBL as an effective alternative to TLR agonists or agonistic Abs to 4-1BB as immunomodulatory component of therapeutic cancer vaccines. Funded in parts by NIH R43 CA 109866 (HS), R43 AI071618 (HS, ESY) R41 CA121665 (HS), KLCRP (HS), KSTC-145-402-22 (HS).

Increased Sensitivity of Radiated Murine Cervical Cancer Tumors to E7 Subunit Vaccine–driven CTL-mediated Killing Induces Synergistic Anti-tumor Activity

The development of therapeutic vaccines has important implications for the treatment of cancer patients. Here we investigate whether human papillomavirus (HPV) E7 subunit vaccines can enhance tumor radioresponse using an established cervical cancer animal model. Radiation plus E7 subunit vaccines improved complete response, cure, and recurrence rates of tumors dramatically compared with single therapy alone. In particular, both components of the E7 subunit vaccines (E7 protein and CpG-oligodeoxynucleotide) were required for the induction of antigen (Ag)-specific cytotoxic T-lymphocyte (CTL) responses and for therapeutic synergy with radiotherapy. Moreover, with combined therapy the radiation dose could be reduced by 16 Gy to achieve an equivalent anti-tumor efficacy to radiation treatment alone. This therapeutic synergy was found to be mediated by CD8(+) CTLs and was concomitant with histological changes (presence of apoptotic bodies and multinucleated giant cells; heavy infiltration of lymphocytes), as determined by in vivo T-cell depletion and histological analysis. Finally, phenotypic changes of radiated tumors and their increased sensitivity to CTL-mediated killing appeared to be responsible for therapeutic synergy. These results show that E7 subunit vaccines act as a potent enhancer of tumor radioresponse and that this is mediated by increased sensitivity of radiated tumors to CTL-mediated killing. This study further suggests that E7-targeted therapeutic vaccines have the potential to improve radiotherapy in patients with cervical cancer.

Eradication of established tumors in mice by a combination antibody-based therapy

Tumor-cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful antitumor immunotherapies. Here we show that induction of tumor-cell apoptosis by an agonistic monoclonal antibody to DR5, the apoptosis-inducing receptor for TNF-related apoptosis-inducing ligand (TRAIL), combined with T-cell activation by agonistic monoclonal antibodies to the costimulatory molecules CD40 and CD137, potently and rapidly stimulated tumor-specific effector CD8+ T cells capable of eradicating preestablished tumors. Primary fibrosarcomas initiated with the carcinogen 3-methylcholanthrene (MCA), multiorgan metastases and a primary tumor containing as many as 90% tumor cells resistant to DR5-specific monoclonal antibody were rejected without apparent toxicity or induction of autoimmunity. This combination therapy of three monoclonal antibodies (trimAb) rapidly induced tumor-specific CD8+ T cells producing interferon (IFN)-gamma in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-gamma in the tumor rejection process. These results in mice indicate that a rational monoclonal antibody-based therapy that both causes tumor-cell apoptosis through DR5 and activates T cells may be an effective strategy for cancer immunotherapy in humans.

Administration Route- and Immune Cell Activation-Dependent Tumor Eradication by IL12 electrotransfer

Injection of DNA via electric pulses into targeted tissues is referred to as electrotransfer. Intratumoral electrotransfer of the IL12 gene is more effective than intramuscular electrotransfer of the same gene in the eradication of established tumors. To understand the underlying immunological mechanisms, T cell infiltration, CTL activity, inhibition of angiogenesis, and transgene expression were analyzed using immunohistochemistry, fluorescence-based CTL analysis, Northern blot, and ELISA. In addition, the therapeutic effects of IL12 gene therapy were determined in immunocompetent, immune-cell-depleted, and immunodeficient mice. We found that intratumoral, but not intramuscular, electrotransfer of the IL12 gene induces CD8+ T cell infiltration, CTL activity, and tumor eradication. Tumor eradication by intratumoral IL12 gene electrotransfer requires both NK and T cells. The absence of either cell type will abrogate the intratumoral IL12 gene therapy-induced tumor eradication. Such a requirement explains why tumors cannot be eradicated by intramuscular electrotransfer of the IL12 gene. Only NK-cell-dependent, and not T-cell-dependent, anti-tumor effects are induced by intramuscular administration. Together, these results suggest that NK cells play an important role in both administration routes, mediating tumor growth inhibition, but T cells are specifically activated by intratumoral IL12 gene electrotransfer and not by the intramuscular route.

Immunity to melanoma mediated by 4‐1BB is associated with enhanced activity of tumour‐infiltrating lymphocytes

4-1BB costimulates T cells to carry out effector functions such as eradication of established tumours. 4-1BB (CD137) is a member of the TNF receptor family, and its triggering by either 4-1BB ligand or antibody ligation induces T-cell activation and growth. We analysed tumour-infiltrating lymphocytes (TIL) in the experimental B16F10 melanoma model to determine the mechanisms involved in 4-1BB-mediated tumour suppression. 4-1BB(+/+) mice survived longer than 4-1BB(-/-) mice, and survival was further prolonged by triggering 4-1BB with an agonistic mAb. The number of metastatic B16F10 colonies in the lung was much greater in 4-1BB(-/-) mice than in their 4-1BB(+/+) littermates. Administration of agonistic anti-4-1BB mAb increased the number of TIL in the tumour masses in the lungs of 4-1BB(+/+) mice. The numbers of CD4(+) T, CD8(+) T and CD11b(+) TIL increased in these mice. Anti-4-1BB mAb induced not only CD8(+) 4-1BB(+) T cells but also a CD8(+) IFN-gamma(+) T-cell population. B16F10 cells from the lungs of anti-4-1BB-treated mice showed enhanced expression of MHC class Iota and IotaIota antigens compared with the same cells from control IgG-treated mice. Thus, the increase in number of CD8(+) T cells and enhanced MHC Iota and IotaIota expression in B16F10 cells that result from augmented IFN-gamma production in response to anti-4-1BB mAb may lead to suppression of tumour growth and metastasis.

Spontaneous elicitation of potent antitumor immunity and eradication of established tumors by administration of DNA encoding soluble transforming growth factor-beta II receptor without active antigen-sensitization.

Since immunity is generally suppressed by immunoregulatory factors, such as transforming growth factor-beta (TGF-beta), interleukin (IL)-10, and vascular endothelial growth factor (VEGF), produced by tumor cells or stromal cells surrounding tumor cells, various kinds of cancer immunotherapy mostly fail to elicit potent antitumor immunity. Herein, we tested whether neutralization of TGF-beta can elicit strong antitumor immune responses and tumor regression in tumor-bearing mice. A plasmid DNA, pcDNA-sTGFbetaR/huIg, encoding a fusion protein consisting of the extracellular domain of TGF-beta type II receptor (TGFbetaRII) and the Fc portion of human IgG heavy chain, was injected through different routes into B6 mice carrying established tumors of E.G7 cells, which consist of the poorly immunogenic tumor cells EL4, transfected with the ovalbumin (OVA) gene. The frequency of OVA-specific cytotoxic T lymphocytes (CTL), in the treated mice. increased resulting in the tumor eradication and relapse-free survival in around 70% of the E.G7-bearing mice. In contrast, administration of mock DNA into E.G7-bearing mice did not elicit tumor-specific immune responses. Therefore, administration of DNA encoding TGFbetaRII allowed tumor-bearing hosts to elicit sufficiently potent antitumor immune responses without requirement of further active antigen-immunization. This strategy seems to be applicable to clinical therapy against cancer, because it is low-cost, safe, and easy to manipulate.

Superior Therapeutic Efficacy of Alphavirus-Mediated Immunization against Human Papilloma Virus Type 16 Antigens in a Murine Tumour Model: Effects of the Route of Immunization

In our efforts to develop a strong, effective immune response against cervical carcinoma and premalignant disease, we study the use of recombinant Semliki Forest virus (SFV) encoding the oncoproteins E6 and E7 from high-risk human papilloma viruses (HPVs). Optimal immunization conditions are required for immunotherapeutic treatment of cervical cancer as it has been postulated that cervical cancer patients are immune-suppressed and/or immunologically tolerant for HPV. We previously generated an optimized construct encoding a fusion protein of HPV16 E6 and E7 and a translational enhancer (enhE6,7). Immunization of mice with SFV-enhE6,7 was shown to induce cytoxic T cell (CTL) responses and resulted in the eradication of established tumours. We now demonstrate, using HPV16-specific MHC class I tetramers, that high pCTL frequencies can be induced. However, this induction is strongly influenced by the route of immunization applied. Whilst in bulk CTL assays, requiring in vitro restimulation, CTL activity can be observed upon s.c., i.p., i.v. and i.m. immunization, detectable pCTL frequencies, without in vitro restimulation, are only induced upon i.m. and i.v. immunization. The route of immunization also strongly influences the dose of viral vector needed to induce CTLs and tumour therapy. As few as 5x10(4) SFV-enhE6,7, primed and boosted i.v., are needed to eradicate tumours in six out of seven mice treated. Furthermore, exponentially growing tumours of approximately 500 mm3 in size were seen to completely resolve and even tumours as large as 1500 mm3 decreased to one-third of their size. Apart from this potency, SFV vectors can safely be used for the expression of oncoproteins such as E6 and E7, since the viral RNA is not integrated in the host genome. Thus SFV-enhE6,7 meets with the criteria that a vaccine against cervical cancer should be safe and induce a very strong, long-lasting CTL response, strong enough to eradicate existing tumours.