Achieve Tumor Eradication Research Articles

Enhancing antitumor immunity and achieving tumor eradication with IL11RA mRNA immunotherapy

Current methods for delivering genes to target tumors face significant challenges, including off-target effects and immune responses against delivery vectors. In this study, we developed a novel approach using messenger RNA (mRNA) to encode IL11RA for local immunotherapy, aiming to harness the immune system to combat tumors. Our research uncovered a compelling correlation between IL11RA expression and CD8 + T cell levels across multiple tumor types, with elevated IL11RA expression correlating with improved overall survival. Examination of the Pan-Cancer Atlas dataset showed a significant reduction in IL11RA expression in various cancer types compared to normal tissue, raising questions about its potential role in tumorigenesis. To achieve efficient in vivo expression of IL11RA, we synthesized two mRNA sequences mimicking the wild-type protein. These mRNA sequences were formulated and capped to ensure effective delivery, resulting in robust expression within tumor sites. Our investigation into IL11RA mRNA therapy demonstrated its effectiveness in controlling tumor growth when administered both intratumorally and intravenously in mouse models. Additionally, IL11RA mRNA treatment significantly stimulated the expansion of CD8 + T cells within tumors, draining lymph nodes, and the spleen. Transcriptome analysis revealed distinct transcriptional patterns associated with T cell functions. Using multiple deconvolution algorithms, we found substantial infiltration of CD8 + T cells following IL11RA mRNA treatment, highlighting its immunomodulatory effects within the tumor microenvironment. In conclusion, IL11RA mRNA therapy presents a promising strategy for tumor regression with potential immunomodulatory effects and clinical implications for improved survival outcomes.

The Use of Bacteria in Cancer Treatment: A Review from the Perspective of Cellular Microbiology.

Cellular microbiology, which is the interaction between harmful microbes and infected cells, is important in the determination of the bacterial infection processes and in the progression of data of different cellular mechanisms. The therapeutic role of bacteria has gained attention since the known methods such as radiation, chemotherapy, and immunotherapy have got drawbacks. Bacteria have demonstrated a favorable impact in treating cancer through eradication of tumors. Bacteria, in cancer treatment, have proven to be promising and have been shown in some of the previous work that it can successfully suppress the growth of tumors. In this paper, we analyzed the difficulties and settlement for using bacteria in cancer therapy as well the mechanisms in which bacteria works in order to achieve tumor eradication. Future works may focus on the use of bacteria along with other treatments in order to achieve effective tumor therapy.

Metformin Mediated PD‐L1 Downregulation in Combination with Photodynamic‐Immunotherapy for Treatment of Breast Cancer

AbstractIntelligent nanomaterials open up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of simple enzymatically cleavable self‐delivery nanoparticles (MA‐pepA‐Ce6 NPs) is developed by conjugating acidic‐sensitive small‐molecule programmed cell death ligand 1 (PD‐L1) inhibitor (Metformin, MET) with photosensitizer (chlorin e6, Ce6) through matrix metalloproteinase‐2 (MMP‐2) cleavable peptide (GPLGVRGDK, pepA). Noticeably, these self‐delivery peptide‐based NPs can circumvent the controversial biosafety facing nanomaterials. Moreover, MA‐pepA‐Ce6 NPs are degraded by overexpressed MMP‐2 in tumor microenvironment (TME) and expose the VRGDK‐Ce6. The exposed VRGDK‐Ce6 shows superior targeting ability towards integrin αvβ3 receptor, ensuring sufficient accumulation and laser‐activated robust antitumor immune effects. Remarkably, the released MET in tumor microenvironment hampers the PD‐L1 expression and augments the antitumor immune response elicited by photodynamics therapy (PDT), thus significantly improving therapeutic outcomes. Overall, this study offers a potential appealing paradigm of synergistic PDT‐triggered immunotherapy by revealing MET‐mediated PD‐L1 downregulation to achieve tumor eradication.

A two-drug combination simulation study for metastatic castrate resistant prostate cancer.

BackgroundProstate cancer often evolves resistance to androgen deprivation therapy leading to a lethal metastatic castrate‐resistant form. Besides androgen independence, subpopulations of the tumor are genetically heterogeneous. With the advent of tumor genome sequencing we asked which has the greater influence on reducing tumor size: genetic background, heterogeneity, or drug potency?MethodsA previously developed theoretical evolutionary dynamics model of stochastic branching processes is applied to compute the probability of tumor eradication with two targeted drugs. Publicly available data sets were surveyed to parameterize the model.ResultsOur calculations reveal that the greatest influence on successful treatment is the genetic background including the number of mutations overcoming resistance. Another important criteria is the tumor size at which it is still possible to achieve tumor eradication, for example, 2‐4 cm large tumors have at best a 10% probability to be eradicated when 50 mutations can confer resistance to each drug.ConclusionOverall, this study finds that genetic background and tumor heterogeneity are more important than drug potency in treating mCRPC. It also points toward identifying metastatic sites early using biochemical assays and/or dPET.

Effective Combination of Innate and Adaptive Immunotherapeutic Approaches in a Mouse Melanoma Model.

Most cancer immunotherapies include activation of either innate or adaptive immune responses. We hypothesized that the combined activation of both innate and adaptive immunity will result in better antitumor efficacy. We have previously shown the synergy of an agonistic anti-CD40 mAb (anti-CD40) and CpG-oligodeoxynucleotides in activating macrophages to induce tumor cell killing in mice. Separately, we have shown that a direct intratumoral injection of immunocytokine (IC), an anti-GD2 Ab linked to IL-2, can activate T and NK cells resulting in antitumor effects. We hypothesized that activation of macrophages with anti-CD40/CpG, and NK cells with IC, would cause innate tumor destruction, leading to increased presentation of tumor Ags and adaptive T cell activation; the latter could be further augmented by anti-CTLA-4 Ab to achieve tumor eradication and immunological memory. Using the mouse GD2+ B78 melanoma model, we show that anti-CD40/CpG treatment led to upregulation of T cell activation markers in draining lymph nodes. Anti-CD40/CpG + IC/anti-CTLA-4 synergistically induced regression of advanced s.c. tumors, resulting in cure of some mice and development of immunological memory against B78 and wild type B16 tumors. Although the antitumor effect of anti-CD40/CpG did not require T cells, the antitumor effect of IC/anti-CTLA-4 was dependent on T cells. The combined treatment with anti-CD40/CpG + IC/anti-CTLA-4 reduced T regulatory cells in the tumors and was effective against distant solid tumors and lung metastases. We suggest that a combination of anti-CD40/CpG and IC/anti-CTLA-4 should be developed for clinical testing as a potentially effective novel immunotherapy strategy.

Antibody-Interferon-Alpha Fusion Protein Therapy for the Treatment of B-Cell Non-Hodgkin Lymphoma: Enhanced ADCC, Inhibition of Proliferation, and In Vivo Eradication of CD20+ Human Lymphomas

Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab.Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice.Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and < 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p < 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression.Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. DisclosuresTimmerman:Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.

Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

B16-derived OVA-expressing melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 cytotoxic T lymphocytes (CTLs) or agonist anti-CD137 (4-1BB) mAb. However when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector function in both transferred OT-1 and endogenous CTLs. This is consistent with higher levels of expression of eomesodermin in CTLs and with confocal microscopy evidence for more efficacious tumor-cell killing. Combined immunotherapy of tumors monitored by intravital live-cell two-photon microscopy reveals persistence of the OT1 CTL-effector phenotype over prolonged periods of time. Anti-CD137 mAb delayed loss of function with focused and confined interaction kinetics of OT-1 CTL with target cells lasting up to ten days post-transfer. The synergy of adoptive T cell therapy and anti-CD137 mAb thus results from in-vivo enhancement of effector functions.

Abstract 925: The c-Met inhibitor MSC2156119J effectively inhibits growth of liver cancer models.

Abstract The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with human malignancies, including hepatocellular carcinomas (HCC). The aim of this study was to evaluate the effect of a novel, highly selective c-Met inhibitor, MSC2156119J (EMD 1214063), in hepatocellular cancer models. MHCC97H is a human HCC cell line co-expressing c-Met and HGF, and exhibiting high metastatic potential. The effect of MSC2156119J on MHCC97H was evaluated in a Balb/c nu/nu murine xenograft model in a subcutaneous and orthotopic setting. Tumor-bearing mice were orally treated with 100 mg/kg MSC2156119J (5 days on/2 days off). In the subcutaneous setting, MSC2156119J resulted in tumor regression in 10/10 mice, achieving tumor eradication in 9/10 mice. In the orthotopic model, MHCC97H tumor fragments proliferate in the liver and invariably metastasize to the lung (100%). MSC2156119J treatment started 7 days after tumor fragment implantation. After 5 weeks of treatment, mice were necropsied and a series of parameters were assessed. Primary tumor size and weight, and circulating Alpha-Feto-Protein levels were significantly lower in mice treated with MSC2156119J compared to controls (p&amp;lt;0.001). Notably, treatment with MSC2156119J resulted in a reduced number of mice with visible lung metastasis (6/9), and correlated with a significantly lower number of metastatic foci in the lungs compared to control mice (p&amp;lt;0.01). To better predict the clinical efficacy of MSC2156119J, 9 patient-derived primary HCC explants models were used. The explants were subdivided into 3 groups, based on their c-Met levels and HGF expression. Each group comprised 3 tumors expressing high, intermediate, and low levels of c-Met and HGF, respectively. Mice were treated with MSC2156119J, sorafenib, or a combination of both. MSC2156119J (100 mg/kg/5 out of 7 days) strongly inhibited tumor growth in 4/9 models (TGD range of 370% to 41%). Analysis of intratumoral c-Met, phospho c-Met and HGF levels indicated that explants with high levels of c-Met and HGF were more sensitive to MSC2156119J than low-expressing models. In 3/4 responsive models, MSC2156119J exhibited a better anti-tumor activity than sorafenib, while sorafenib was more efficacious in a model characterized by intermediate c-Met/HGF expression. MSC2156119J was not efficacious in models exhibiting low or no signs of c-Met signalling. When used in combination, MSC2156119J did not enhance the activity of sorafenib. MSC2156119J monotherapy was well tolerated while sorafenib alone and in combination with MSC2156119J induced significant body weight loss. Overall, these data indicate that MSC2156119J may be a valuable therapeutic option for liver cancers with high levels of c-Met expression/activation. Citation Format: Friedhelm Bladt, Andree Blaukat, Dieter Dorsch, Manja Friese-Hamim, Michael Meyring, Oliver Schadt. The c-Met inhibitor MSC2156119J effectively inhibits growth of liver cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 925. doi:10.1158/1538-7445.AM2013-925

Abstract 2835: Combined stimulation of TLR9 and 4.1BB receptors enhances Trp2 peptide-mediated B16 melanoma regression by increasing Ag-specific CTL activity and infiltration to tumor sites.

Abstract We previously reported that intratumoral electroporation (IT-EP) with IL-12 cDNA (IT-EP/IL12) led to the eradication of established B16 melanoma tumors in mice. Tumor eradication required the participation of CD8+ T cells, but not CD4+ T cells and NK cells. IT-EP/IL12 induced antigen-specific CD8+ T cell responses against the immunodominant Trp2180-188 epitope and generated a systemic response, resulting in significant therapeutic effects against distal, untreated tumors. In this study, we observed that when Trp2180-188 self-peptides were combined with the TLR9 agonist, CpG-oligodeoxynucleotide (ODN) for antitumor immune therapy, self-tolerance was broken, giving rise to induction of Trp2-specific CTL responses and some antitumor activity against established melanoma (7 mm in mean diameter). A combination of anti-4.1BB antibodies with Trp2 peptides plus CpG-ODN increased the antitumor regression rate from 0 to 75%. This effect was concomitant with greater induction of Ag-specific CD8+ CTLs and their infiltration into the tumor sites, highlighting the importance of combined stimulation of TLR9 and 4.1BB for achieving tumor eradication. These findings may have implications for designing peptide-based therapeutic vaccines for cancer-patients. Citation Format: Jeong-Im Sin, Hun Mo Ryoo. Combined stimulation of TLR9 and 4.1BB receptors enhances Trp2 peptide-mediated B16 melanoma regression by increasing Ag-specific CTL activity and infiltration to tumor sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2013-2835

Combined stimulation of TLR9 and 4.1BB augments Trp2 peptide vaccine-mediated melanoma rejection by increasing Ag-specific CTL activity and infiltration into tumor sites

Peptide vaccines are a clinically applicable therapy shown to be effective in tumor control. In this study, Trp2 peptides plus CpG-oligodeoxynucleotide treatment was found to induce Ag-specific IFN-γ and CD8+CTL responses, and antitumor activity against large established melanoma (tumor size, 7mm). A combination of anti-4.1BB antibodies with Trp2 peptides+CpG-oligodeoxynucleotide increased the antitumor cure rate from 0% to 75%. This effect was concomitant with greater induction of Ag-specific CD8+CTLs and their infiltration into the tumor sites, highlighting the importance of combined stimulation of TLR9 and 4.1BB for achieving tumor eradication. These findings may have implications for designing peptide-based therapeutic vaccines for cancer-patients.

Abstract 5273: XenTech patient-derived xenograft (PDX) panels: a clinically relevant platform for drug efficacy, target validation and predictive biomarker discovery studies

Abstract Despite considerable efforts in understanding the biology and genetics of cancer, most currently available treatments fail to achieve tumor eradication in the majority of patients. Key to more effective therapies is adequate disease classification and subsequent patient stratification. In addition, it is important to understand the mechanisms of drug-response or resistance and identify novel targets amenable to therapeutic intervention. It is increasingly recognized that at the preclinical stage, testing therapeutic strategies and validating target relevance in more predictive models closely mimicking clinical disease such as patient derived xenografts (PDXs), may translate into improved clinical efficacy and lower rate of drug attrition. XenTech collection of over 120 runing PDX models is one of the largest in the world. PDX models were established by grafting post-surgery human tumor fragments in the interscapular region of immunodeficient mice. These deeply characterized PDX models can be used for in vivo preclinical assays. Such preclinical platform is a reliable surrogate of patient cohorts and can address several aims: 1. Evaluate tumor response to treatment. PDXs can be subjected to parallel evaluation of tumor response to various treatment protocols. Drug-response profile is linked to tumor histotype and molecular features in order to identify predictive markers of drug response to assist treatment choice. 2. Assess treatment-driven tumor eradication. The ability of a treatment to induce complete tumor response is assessed by monitoring tumor regression over a long period. Most tumors, despite complete macroscopic regression, are still present as latent microscopic nodular islands that may give rise to tumor recurrence. Molecular characterization of tumor foci responsible for tumor relapse may be performed to identify genes/pathways involved in residual tumor cell survival, which may provide new diagnostic and/or therapeutic targets for designing novel adjuvant treatment strategies. 3. Development of bioluminescent metastatic models to study the mechanisms of tumor invasion and to test anti-metastatic therapy. 4. Non-invasive molecular imaging technology to monitor tumor metabolism, vascularization and apoptosis. 5. Constitution of preclinical panels of rare malignancies to obtain phase II-like tumor cohorts. Development of new therapies for rare tumors is rendered difficult by the unavailability of patient cohorts wide enough to set up robust clinical trials. To assist the clinical need, these panels would allow the evaluation of new and more efficient therapies. We describe here in detail our PDX collection and illustrate how it represents a powerful tool to identify preferential therapeutic options for patients by exploring and improving anti-cancer therapeutic strategies. 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 5273. doi:1538-7445.AM2012-5273

Immunology and Gene Therapy: Shoulder to Shoulder Into the Fray: International Symposium: Critical Frontiers Between Immunity and Gene Therapy Pamplona, Spain, 27–28 October 2009

Functions of the immune system are critical for the outcome of gene therapy. Immunologists, virologists, and gene therapists met in Pamplona, Spain, in October 2009 to present and analyze common frontiers in these fields under the auspices of the Ramon Areces Foundation (http://www.fundacionareces.es). Antibodies and cell-mediated immune mechanisms can be either friends or foes in gene therapy. A form of Murphy’s law seems to apply: “If an immune response is to be induced, it will usually be weak. If an immune response is to be avoided, it will be almost always neutralizing.”

Limitations of Adenoviral Vector-Mediated Delivery of Gold Nanoparticles to Tumors for Hyperthermia Induction

Novel combinatorial treatment strategies are desired to achieve tumor eradication. In this regard, nanotechnology and gene therapy hold the potential to expand the available tumor treatment options. In particular, gold nanoparticles (AuNPs) have been utilized for hyperthermic tumor cell ablation. Similarly, adenoviral (Ad) vectors have been utilized for targeting, imaging, and cancer gene therapy. Thus, to combine AuNP-mediated hyperthermia with Ad vector-based gene therapy, we have previously coupled AuNPs to Ad vectors. Herein we tested the capability of these AuNP-coupled Ad vectors for hyperthermic tumor cell ablation. Towards this end, we compared absorption characteristics of different sized AuNPs and determined that in our system 20 nm diameter AuNPs are suitable for laser induced hyperthermic tumor cell killing. In addition, we observed that AuNPs outside and inside the cell contribute differentially towards hyperthermia induction. Unfortunately, due to the limitation of delivery of required amounts of AuNPs to cells, we observed that AuNP-coupled Ad vectors are unable to kill tumor cells via hyperthermia. However, with future technological advances, it may become possible to realize the potential of the multifunctional AuNP-coupled Ad vector system for simultaneous targeting, imaging, and combined hyperthermia and gene therapy of tumors.

In Vivo Eradication of a Rituximab-Resistant Human CD20+ B Cell Lymphoma by Rituximab-CpG Oligodeoxynucleotide Conjugate Is Mediated by Natural Killer Cells and Complement.

Abstract 723The anti-CD20 monoclonal antibody rituximab exerts its anti-tumor effects against B cell non-Hodgkin's lymphoma (NHL) via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG ODN) are potent activators of ADCC and T cell immunity, and have been studied for anti-NHL effects when administered by systemic or intratumoral (i.t.) routes. We have used an aggressive human CD20-expressing syngeneic murine B cell lymphoma (38C13-huCD20) that is resistant to single agent rituximab to study techniques for improving antibody efficacy. We recently reported that combining rituximab with i.t. (but not systemic) CpG promotes eradication of 7-day established rituximab-resistant 38C13-huCD20 lymphoma from 42% of mice (D. Betting et al, J. Immunotherapy 2009). Six doses of i.t. CpG (50 μg days 7, 8, and 9 and 25 μg days 11, 13, and 15) were required to achieve tumor eradication in this setting. Both natural killer cells and complement participated in the cure of tumors by rituximab plus i.t CpG, by increasing tumor cell sensitivity to ADCC and complement lysis, and by augmenting the cytotoxicity of ADCC effectors. To overcome the need for repeated i.t. injections, eliminate the need for an accessible tumor site, and allow for more direct targeting of CpG to tumor cells, we chemically linked CpG to rituximab using a cleavable linker. The conjugate retained both antibody binding activity to human CD20 and potent CpG biologic activity, demonstrated via upregulation of costimulatory molecules on cultured lymphoma cells and activation of bone marrow-derived dendritic cells. Just 2 injections of rituximab-CpG conjugate (containing 50 μg CpG, days 7 and 9) achieved eradication of 7-day established tumors from 100% of mice, while injection of rituximab-control ODN or trastuzumab-CpG conjugates had no therapeutic effects. Treatment with equivalent doses of rituximab plus i.v. or i.t. CpG prolonged survival slightly, but did not result in tumor eradication. In vivo depletion of NK cells or complement nearly eliminated tumor eradication effects. In contrast, in vivo depletion of macrophages, or CD4+ or CD8+ T cells had no significant effects on tumor eradication by rituximab-CpG, indicating that adaptive T cell immunity did not contribute to elimination of tumor during rituximab-CpG conjugate therapy. As CpGs can stimulate leukocytes to secrete alpha interferon (αaIFN), which can inhibit the growth of B cell lymphomas, we sought to determine whether αaIFN played a significant role in tumor control after rituximab-CpG therapy. 38C13-huCD20 tumor cells rendered deficient in αaIFN receptor (IFNAR) by lentiviral transduction with shRNA were nearly as sensitive to rituximab-CpG as wild-type IFNAR+ 38C13-huCD20 cells, indicating no role for direct αaIFN tumor cytotoxicity in eradication of 38C13-huCD20. In conclusion, natural killer cells and complement, but not T cells, macrophages, or αaIFN, are critical to the efficacy of rituximab-CpG conjugate in efficiently eradicating an established human CD20+ B cell lymphoma that is fully resistant to single-agent rituximab. Further pre-clinical studies of anti-CD20-CpG conjugates against B cell lymphomas are thus warranted. Anti-CD20-CpG conjugates may represent a novel treatment modality for human NHL. Disclosures:No relevant conflicts of interest to declare.

Intratumoral But Not Systemic Delivery of CpG Oligodeoxynucleotide Augments the Efficacy of Anti-CD20 Monoclonal Antibody Therapy Against B Cell Lymphoma

The anti-CD20 monoclonal antibody rituximab (Rituxan) has become a mainstay in the treatment of B cell non-Hodgkin lymphomas. The mechanisms of action for rituximab include antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Combination of anti-CD20 antibodies with immunostimulatory agents may improve their efficacy via enhancement of one or more of these mechanisms. Toll-like receptor 9 agonist CpG oligodeoxynucleotides administered systemically have been studied in clinical trials with and without rituximab. However, recent data suggest that intratumoral (IT) delivery of CpG has advantages in the treatment of tumors. Using a syngeneic murine B cell lymphoma line expressing human CD20, we found that IT, but not systemically administered CpG significantly improved the efficacy of rituximab against 7-day established tumors. Rituximab plus IT CpG could eradicate tumors from 42% of mice, whereas systemically administered CpG, with or without rituximab, did not achieve tumor eradication. Both natural killer cells and complement participated in the cure of tumors by rituximab plus IT CpG, apparently by increasing tumor cell sensitivity to complement and ADCC lysis, and by augmenting the cytotoxicity of ADCC effectors. No role for T cells in mediating tumor eradication was demonstrated in this model. These results suggest that previous clinical trials in B cell lymphoma combining systemic administration of CpG with rituximab may have employed suboptimal routes of CpG delivery. Future trials combining IT CpG with anti-CD20 antibodies or the antibody-mediated targeting of CpG directly to the sites of B cell lymphoma may thus be warranted.

Photocatalytic Generation of Oxygen Radicals by the Water-Soluble Bacteriochlorophyll Derivative WST11, Noncovalently Bound to Serum Albumin

Light-induced radical generation is the hallmark of fundamental processes and many applications including photosynthesis and photodynamic therapy (PDT). In this manuscript, we present two novel observations made upon monitoring light-induced generation of reactive oxygen species (ROS) in aqueous solutions by WST11, a water-soluble derivative of the photosynthetic pigment Bacteriochlorophyll a (Bchl). Using a host of complementary experimental techniques including time-resolved spectroscopy at the subpicosecond to the millisecond range, ESR spectroscopy, electrochemistry, spectroelectrochemistry, oximetry, and protein mass spectroscopy, we first show that in aqueous solutions WST11 generates only superoxide (O(2)(-*)) and hydroxyl (OH*) radicals with no detectable traces of singlet oxygen. Second, we show that WST11 makes a noncovalent complex with human serum albumin (HSA) and that this complex functions as a photocatalytic oxidoreductase at biologically relevant concentrations enabling approximately 15 cycles of electron transfer from the associated HSA protein to molecular oxygen in the solution. These findings rule out the paradigm that porphyrin and chlorophyll based PDT is mainly mediated by formation of singlet oxygen, particularly in vascular targeted photodynamic therapy (VTP) with sensitizers that undergo photoactivation during circulation in the plasma, like [Pd]-Bacteriopheophorbide (WST09, Tookad). At the same time, our findings open the way for new design paradigms of novel sensitizers, since O(2)(-*) and OH* radicals are well-recognized precursors of important pathophysiological processes that can be activated for achieving tumor eradication. Moreover, the finding that promiscuous protein scaffolds become sinks for holes and electrons when holding light-activated pigments provides a new insight to the evolution and action mechanism of natural light activated oxidoreductases (such as photosynthetic reaction centers) and new guidelines for the preparation of synthetic-light converting machineries.

Mesenchymal Stem Cell Differentiation Markers Shared with Soft Tissue Sarcomas

Malignant tumors are hypothesized to harbor small populations of self-renewing cancer stem cells. Targeting these cells may be the decisive step to overcome treatment resistance and achieve tumor eradication in cancer patients. Advanced soft tissue sarcomas (STS) are rare tumors with a dismal prognosis and a small number of systemic treatment options. STS may originate from mesenchymal stem cells (MSC); the latter have mainly been isolated from adult bone marrow (BM) as non-hematopoietic, self-renewing cells whose in vitro progeny comprises osteoblasts, chondroblasts, myocytes, and adipocytes. While in vitro expression profiles of MSC have been investigated extensively, the in vivo counterparts of MSC are still hypothetical. To target rare human cell BM populations including MSC, an exclusive antibody panel was developed. The target antigens include platelet-derived growth factor receptor-β (CD140b), HER-2/erbB2 (CD340), the recently described W8B2 antigen as well as several surface antigens identified by novel antibodies. To define the expression pattern of MSC-markers in STS, three STS cell lines were tested for expression of these antigens. In addition, snap-frozen primary STS sections were analyzed by immunohistochemistry using the same antibody panel. All cell lines revealed expression of selected markers including CD340, W8B2, and CD140b. Several MSC markers were restricted to a subpopulation of cells. In addition, leiomyosarcoma cells displayed a different expression pattern as compared to liposarcoma and Ewing's sarcoma cells. Results of immunohistochemistry analysis of primary leiomyosarcoma tumor samples correlated strongly with expression patterns established by FACS analysis. However, important cytoarchitectural features regarding selected markers were revealed by immunohistochemistry: while primary leiomyosarcomas displayed uniform expression of W7C6, HEK3D6, CD10, and CD318, other markers such as CD34, W5C5, and 57D2 were expressed by tumor endothelia only. Moreover, a population of perivascular tumor cells was found to express the MSC-markers W4A5, W8B2, CD140b, W3D5, and W5C4. Novel MSC-markers are expressed by subpopulations in STS cell lines as well as in primary sarcoma tissue. Further studies on the functional significance of these phenotypical studies are underway and may help to identify novel specific targets recognizing the self-renewing STS-compartment.

Adjuvants targeting innate and adaptive immunity synergize to enhance tumor immunotherapy

Because of mechanisms of self-tolerance, many tumor-specific CD8 T cells exhibit low avidity for tumor antigens and would benefit from strategies that enhance their numbers and effector function. Here we demonstrate that the combined use of two different types of immune adjuvants, one that directly targets the CD8 cell, IL-2/anti-IL-2 mAb complexes, and one that targets the innate immune system, poly(I:C), can achieve this goal. Provision of IL-2/mAb complexes was found to enhance the activation and effector function of low-avidity tumor-specific T cells, yet this was insufficient to achieve tumor eradication. The addition of poly(I:C) further increased the accumulation of granzyme B-expressing effectors within the tumor and resulted in tumor eradication. This strategy presents many of the benefits of whole-body irradiation, including the provision of high levels of homeostatic cytokines, enhanced expansion of effector cells relative to regulatory T cells, and provision of inflammatory cytokines, and is therefore likely to serve as a strategy for both tumor vaccines and adoptive immunotherapy of cancer.

Abstract 4908: Antennapedia "trojan" peptide delivers p21 protein resulting in tumor eradication

Abstract The purpose of this study is to utilize the “trojan horse” approach to deliver an effector function to cancer cells in vitro and in vivo. Many cell-translocating trojan peptides have been studied in the context of therapeutic protein delivery, including the homeodomain region of the Drosophila Antennapedia protein. The mechanism of transduction of this peptide relies on the presence of basic aminoacids arranged in helical conformations, which are important for lipid interactions and for penetration of the membrane. The tumor-suppressor protein p21 is important in many key cell division processes including G1 arrest leading to apoptosis of damaged cells. Restoring p21 protein to tumor cells lacking functional p21 could lead to tumor cells regaining higher chemosensitivity and the ability to die upon genotoxic insult. Our experiments investigate the tumor inhibitory and therapeutic potential of a fusion protein comprising of the ‘trojan’ peptide Antennapedia and p21. The effects of administration of Antennapedia-p21 on the growth of cultured cancer cells and on tumors implanted into nude mice were evaluated. In vitro studies showed that nuclear localization of p21 protein could be achieved using the Antennapedia carrier. The efficacy of this treatment on cells was also determined in combination with conventional chemotherapy. Treatment with Antp-p21 induced cell-cycle arrest leading to the inhibition of tumor cell growth. Intra-venous administration of the fusion protein to tumor-bearing mice inhibited tumor growth and prolonged overall survival. Furthermore, co-administration of the fusion protein with conventional chemotherapeutic agents resulted in enhanced growth inhibitory effects, tumor eradication in 40% of animals and significant reduction in tumor burden in 100% of animals. In conclusion, our findings show that we can utilize the “trojan horse” approach to deliver an effector function to cancer cells in vitro and in vivo and achieve tumor eradication. The efficacy of our anti-tumor growth modality in combination with conventionally used anti-neoplastic medication suggests that it can be considered as a promising therapeutic drug for the management of a wide range of carcinomas.