Marnix L. Bosch

Current methods for loading dendritic cells with tumor antigen for the induction of antitumor immunity

The immunotherapy of cancer is predicated on the belief that it is possible to generate a clinically meaningful antitumor response that provides patient benefit, such as improvement in the time to progression or survival. Indeed, immunotherapeutics with dendritic cells (DC) as antigen-presenting delivery vehicles for cell-based vaccines have already improved patient outcome against a wide range of tumor types (1–9). This approach stimulates the patients own antitumor immunity through the induction or enhancement of T-cell immunity. It is generally believed that the activity of cytotoxic T lymphocytes (CTL), the cells directly responsible for killing the tumor cells in vivo, are directed by DC. Therefore, the goal of many current designs for DC-based vaccines is to induce strong tumor-specific CTL responses in patients with cancer. In practice, most studies for DC-based cancer vaccine development have focused on the development of methods that can effectively deliver exogenous tumor antigens to DC for cross-priming of CD8+ T cells through the endogenous MHC class I processing and presentation pathway (10). To date, many methods have been developed or evaluated for the delivery of defined and undefined tumor antigens to DC. This review provides a brief summary on these methods, the techniques used in these methods, as well as the advantages and disadvantages of each method.

Interferon-γ Added During Bacillus Calmette-Guerin Induced Dendritic Cell Maturation Stimulates Potent Th1 Immune Responses

Dendritic cells (DC) are increasingly prepared in vitro for use in immunotherapy trials. Mature DC express high levels of surface molecules needed for T cell activation and are superior at antigen-presentation than immature DC. Bacillus Calmette-Guerin (BCG) is one of several products known to induce DC maturation, and interferon (IFN)-γ has been shown to enhance the activity of DC stimulated with certain maturation factors. In this study, we investigated the use of IFN-γ in combination with the powerful maturation agent, BCG. The treatment of immature DC with IFN-γ plus BCG led to the upregulation of CD54, CD80, and CD86 in comparison with BCG treatment alone. In MLR or recall immune responses, the addition of IFN-γ at the time of BCG-treatment did not increase the number of antigen-specific T cells but enhanced the development of IFN-γ-producing Th1 cells. In primary immune responses, on the other hand, BCG and IFN-γ co-treated DC stimulated higher proportions of specific T cells as well as IFN-γ secretion by these T cells. Thus the use of IFN-γ during BCG-induced DC maturation differentially affects the nature of recall versus naïve antigen-specific T-cell responses. IFN-γ co-treatment with BCG was found to induce IL-12 and, in some instances, inhibit IL-10 secretion by DC. These findings greatly enhance the potential of BCG-matured dendritic cells for use in cancer immunotherapy.

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNγ release from an M1-specific T-cell line, expansion of M1 peptide-specific Vβ17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.

Abstract 2491: Treatment with tumor lysate-pulsed autologous dendritic cells prolongs survival in patients with recurrent glioblastoma multiforme

Background The survival rate in recurrent Glioblastoma multiforme (rGBM) patients has not meaningfully changed in the past several decades. We have treated two cohorts of rGBM patients, one consisting of patients with early progression, and one of patients with progression following several cycles of adjuvant temozomolide chemotherapy, with autologous dendritic cells pulsed with autologous tumor cell lysate (DCVax®-L). Such treatment is intended to activate the immune system against the tumor cells, so that the ensuing immune attack may delay progression and time to death. Methods Disease progression in patients with GBM was determined through MRI. Progression needed to be determined by independent review on 2 consecutive scans for early progressor classification to avoid enrolling patients with pseudoprogression. Assessment of progression was done using modified RANO criteria. Results Nineteen (19) patients diagnosed with GBM were determined to have recurrence immediately following radiation therapy with concomitant temozolomide chemotherapy. Median overall survival in this cohort from initial GBM diagnosis is 15.1 months (95% CI: 10.5-17.2), and the range is 8.1->31 months. A literature search revealed 6 publications with comparable populations of patients. The table below demonstrates that these patients typically have a life expectancy of 8-10 months. A second cohort of patients with rGBM consisted of 8 patients with recurrence following several adjuvant temozolomide treatment cycles. Median OS for these patients is 14.7 months from the time of surgery for first recurrence. The comparator for this cohort is derived from Friedman et al. (2009), who reported mOS of 8.7-9.3 months from the time of first recurrence for rGBM patients treated with Bevacizumab with or without Irinotecan. Results and Conclusion These results suggest that treatment with DCVax-L can extend survival by 5 months or more in patients with rGBM. Citation Format: Marnix Bosch, Robert Prins, Linda Liau. Treatment with tumor lysate-pulsed autologous dendritic cells prolongs survival in patients with recurrent glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2015-2491

A novel series of conferences tackling the hurdles confronting the translation of novel cancer immunotherapies

While there has been significant progress in advancing novel immune therapies to the bedside, much more needs to be done to fully tap into the potential of the immune system. It has become increasingly clear that besides practical and operational challenges, the heterogeneity of cancer and the limited efficacy profile of current immunotherapy platforms are the two main hurdles. Nevertheless, the promising clinical data of several approaches point to a roadmap that carries the promise to significantly advance cancer immunotherapy. A new annual series sponsored by Arrowhead Publishers and Conferences aims at bringing together scientific and business leadership from academia and industry, to identify, share and discuss most current priorities in research and translation of novel immune interventions. This Editorial provides highlights of the first event held earlier this year and outlines the focus of the second meeting to be held in 2013 that will be dedicated to stem cells and immunotherapy.

Correction: First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma [J Transl Med., 16, (2018) (142)] DOI: 10.1186/s12967-018-1507-6

Following publication of the original article [1], the authors reported an error in the spelling of one of the author names. In this Correction the incorrect and correct author names are indicated and the author name has been updated in the original publication. The authors also reported an error in the Methods section of the original article. In this Correction the incorrect and correct versions of the affected sentence are indicated. The original article has not been updated with regards to the error in the Methods section.

Cytokines Produced by Dendritic Cells Administered Intratumorally Correlate with Clinical Outcome in Patients with Diverse Cancers

Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice. Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection. Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01). Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes. Clin Cancer Res; 24(16); 3845–56. ©2018 AACR.

Abstract B005: Cytokine production by intratumorally administered activated dendritic cells correlates with survival in a Phase I clinical trial in diverse cancers

Background: Activated, autologous dendritic cells (aaDC) can be used to induce anti-tumor immune responses. A unique method of applying aaDC is through intratumoral injection, where the tumor cells serve as the source of antigen required for an adaptive anti-tumor response. A local effect may also occur as a result of cytokine production by the injected DC which makes the tumor more susceptible to a pre-existing or an induced immune attack. Methods: Forty patients with locally advanced or metastatic solid tissue cancers were treated in a dose escalation trial in which aaDC were injected percutaneously under image guidance into a single tumor. Subjects had a median of 3 tumors (range 1 - 5) and had received an average of 3.1 prior treatments. To generate the aaDC, autologous monocytes were converted ex vivo into DC which were then activated. All batches of DC were released based on pre-specified criteria which included immunophenotyping and a T cell-stimulation assay, as well as sterility and endotoxin levels. Cytokine levels produced by the activated DC during manufacturing were measured and patient outcomes were correlated to these expression levels. Results: All three doses levels were well tolerated. The main adverse events related to treatment were grade 1 and 2 fevers. Twenty-one patients achieved stable disease (SD) 8 weeks after initiating treatment, and this was found to correlate with survival (p = 0.01). Levels of certain cytokines, such as such IL-8 and IL-12 p40, and TNFα were substantially elevated in vitro and IL-8 and IL-12 p40 production were predictive of survival (p = 0.001 and p = 0.008 resp.). TNFα levels also correlated with SD at week 8 (p = 0.01). More than 70% of patients tested were found to have significant T cell responses, and/or de novo or significantly enhanced PD-L1 expression in the tumor post treatment, with a trend towards improved survival (p = 0.1). Conclusions: Study outcomes such as stabilization of disease and survival correlated with high DC cytokine levels, in the absence of meaningful toxicity. The DCVax treatment may be mediated through direct cytotoxic effects, as well as modulation of the tumor microenvironment to increase tumor infiltration by T cells, and attraction of inflammatory cells to the tumor. The development of PD-L1 expression likely reflects an induced immune response. Citation Format: Vivek Subbiah, Ravi Murthi, Robert Prins, Kyle Hendricks, Chitra Hosing, Lori Noffsinger, Mary McGuire, Robert Brown, Aung Naing, David Hong, Siqing Fu, Anthony Conley, Indreshpal Kaur, Sarah Campion, Marnix Bosch. Cytokine production by intratumorally administered activated dendritic cells correlates with survival in a Phase I clinical trial in diverse cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B005.

Safety, feasibility and functionality of activated autologous dendritic cells for intratumoral injection in solid tumors: a Phase I clinical trial

Meeting abstracts Dendritic cells (DC) are proficient in initiating adaptive immune responses, through the uptake and subsequent presentation to the immune system of antigenic compounds. In preclinical studies, activated DC (aDC; DCVax®-Direct) were shown to be superior to immature DC in clearing

Abstract 2499: Clinical and immunopathological effects following Image-guided intratumoral injection of activated, autologous dendritic cells in patients with advanced solid cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Preclinical models have shown that activated DCs can effectively clear both injected (local) and non-injected (distal) inoperable tumor lesions. Based on this strong rationale a Phase I trial was designed to exploit this finding in late stage solid tumors. Methods: We designed a phase I study to determine the safety of intratumoral injection of activated DC by administering the product repeatedly until disease progression or unacceptable toxicity, in advanced cancers. Endpoints included maximum tolerated (MTD), dose limiting toxicities (DLT), safety, and immune response criteria (iRC) and RECIST. The treatment consisted of image-guided intratumoral (IT) injections of autologous, activated dendritic cells (DCVax-Direct,). Injections were given on days 0, 7 and 14, followed by booster immunizations at weeks 4, 8 and 16. Three dose levels, at 2 million, 6 million or 15 million DC per injection, were tested. Concomitant, serial biopsies were performed at the time of the vaccination in the absence of toxicity /progression. Systemic immune responses were tracked through evaluation of T cell subsets and cytokines in circulation, and through T cell receptor (TCR) sequencing in the tumor tissue and in the periphery. Results: To date, 40 patients (men, n = 18; women, n = 22), median age 53.7 (range 30 - 73) years, median of 3.1 (1 - 6) prior therapies (including 1 patient with prior immune therapy) were enrolled. Seventeen patients were treated at the 2 million dose level, 19 at the 6 million dose level, and 3 at 15 million. The MTD has not been reached and there were no dose limiting toxicities. Two patients experienced Grade 3/4 drug related toxicities : one case of fever and dehydration and one case of systemic inflammatory response syndrome. Patterns of immunological reactivity were assessed by pathological scoring on tumor biopsies for 29 patients, and included increasing necrosis in 62% of patients and emergence or amplification of infiltrating T cells in 55%. In-depth study was carried out in a patient with therapy-refractory, de-differentiated liposarcoma whose imaging at 12 weeks post initial injection revealed necrosis in the primary tumor and stable lung metastases. Biopsies demonstrated an intra-tumoral inflammatory response consisting of lymphocytes, macrophages and TIA-1 expressing cells, suggesting cytolytic activity. TCR sequencing revealed the presence of shared TCR sequences in the tumor as well as in circulation, demonstrating a systemic anti-tumor response. Analysis of T cell subsets in circulation demonstrated normalization of the CD4/CD8ratio. Conclusion: Immunotherapy with partially activated DCs injected IT demonstrate early signals of immune reactivity even in late stage patients with cancer. Preliminary data support the generation of anti-tumor effects following IT injection of the partially activated, autologous DC’s. Citation Format: Vivek Subbiah, Ravi Murthy, David S. Hong, Robert E. Brown, Robert Prins, Chitra Hosing, Mary McGuire, Aung Naing, Siquing Fu, Tina Chou, Quan Lin, Richard P. Guevarra, Anthony Conley, Indreshpal Kaur, Funda Meric-Bernstam, Marnix Bosch. Clinical and immunopathological effects following Image-guided intratumoral injection of activated, autologous dendritic cells in patients with advanced solid cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2015-2499