Clinical Trials of Dendritic Cell Therapies for Cancer: Biotech's Bumpy Road to the Market

Abstracts

Cellular Immunotherapy for T Cell Lymphoma: Advantages of Maturationally Synchronized, Physiologically Induced DC
Richard L. Edelson, MD, Yale School of Medicine

The cumulative clinical experience with Extracorporeal Photochemotherapy (ECP), an FDA-approved cellular immunotherapy, supports the premise that dendritic antigen presenting cells (DC) can be potent immune master switches, even in the context of advanced T cell mediated disease.  On the basis of multiple clinical studies in cutaneous T cell lymphoma (CTCL), as well as in autoreactive disease (acute and chronic graft versus host disease and transplanted heart and lung rejection), ECP is currently performed in more than 500 university medical centers, divided equally between Europe and the United States.  CTCL and transplantation reactions were selected as the clinical targets, because responses to therapy can be readily assessed via real-time clinical and laboratory hard data determinants.  Since our group introduced ECP in 1988, as an immunization procedure for naturally lethal advanced CTCL, it has been administered more than 1 million times, to more than 30,000 patients.  For CTCL, the 2hr treatment sequentially involves:  (1) initiation of apoptosis of pathogenic T cells, by their ex vivo exposure in a flow device, to ultraviolet A (UVA) activated 8-methoxypsoralen (8-MOP); (2) induction of monocyte-to-DC maturation by passage of leukapheresed blood, as a thin film, through a plastic chamber; (3) loading of new DC with tumor antigens via internalization of apoptotic CTCL cells; (4) immunizing intravenous return of the armed DC vaccine.  The ex vivo routing of blood through the ECP device efficiently directs a substantial minority of passaged monocytes to enter the DC maturational pathway, while globally activating the large majority of the processed monocytes to express cell surface attributes of active antigen presenting cells.   The transcriptome signature of the ECP-processed monocytes is distinctive, marked by the consistent activation of nearly 500 genes, including many with known immunologic import. The initiating trigger of this phenomenon is transient adherence of the extracorporeally passaged monocytes to ECP device-immobilized autologous platelet p-selectin. Preliminary evidence that a subpopulation of the induced DC express high levels of GILZ gene activation, a feature that typifies down-regulatory DC, after exposure to relatively high levels of UVA, provides a potential explanation of ECP’s immunologically bidirectional clinical effects.  Recognition of the central role of activated platelets in directing monocytes into the DC maturational pathway, coupled with awareness that photoactivated 8-MOP skews maturing DC into an immuno-regulatory mode, has permitted logical design clinical trials for a broader range of immunogenic cancers.  Since UVA-activated 8-MOP is not involved in the DC induction step, but instrumentally causes gradual apoptosis of malignant cells which can then be DC-processed as a source of immunogenic tumor antigens, trials can be designed to potentially maximize anti-cancer vaccination by controlling where, in the cell processing sequence, UVA is discretely applied.  Also, a newly developed scalable mouse-to-man miniaturized device can enable both informative experimental animal studies and tuning of identified variables in human trials.
 

Early Dendritic Cell Trials at Stanford: Setting the Stage for Sipuleucel-T
Edgar G. Engleman, MD, Stanford University School of Medicine

Efforts to overcome immune tolerance to tumors have focused in part on dendritic cells (DCs), based on their unmatched ability to stimulate both the innate and cognate arms of the immune system. In the early 1990s, after developing relatively simple methods to isolate and activate human blood DCs, we decided to explore the potential utility of these cells in tumor immunotherapy. Our first DC vaccine trial was performed in patients with B cell lymphoma who had failed conventional treatment.  In that trial, DCs from the patients were loaded with tumor specific immunoglobulin and matured in vitro before being returned to the patients.  Despite their advanced disease and the relatively small number (<5x106) of DCs infused, all four vaccinated patients developed T cell responses against tumor Ig and two of the patients went into complete clinical remission. Following that pilot trial we studied DC vaccination in prostate cancer, multiple myeloma and colorectal cancer, with encouraging results. Our technology was licensed by Dendreon Corporation, which used it to develop a novel DC vaccine (Sipuleucel-T) for metastatic prostate cancer. In 2010 this vaccine was approved by the FDA, based on the demonstration of a significant survival benefit in patients participating in controlled clinical trials.  However, more potent and less expensive approaches to DC based immunotherapy are needed.  One approach that has shown promise in animal models is to load and activate DCs, in vivo. This strategy is beginning to be evaluated in clinical trials and has the potential to lead to the next generation of DC vaccines.

Danger Signals in Cancer Immune Therapy with Dendritic Cells: Experience and First Trends from a Randomized Clinical Trial
Thomas Felzmann, MD, Activartis Biotech GmbH, Austria

Dendritic cells (DC) respond to the notion of danger that comes in different guises to initiate an activation or differentiation process, conventionally referred to as maturation. Binding of microbial pattern molecules such as lipopolysaccharides (LPS) to Toll-like receptors (TLR) on DCs signal danger. Upon TLR engagement, DCs assume a potent pro-inflammatory and immune stimulatory phenotype characterized by the release of IL-12 for approximately one day. IL-12 secreting DCs trigger robust immune responses dominated by type 1 helper T- lymphocytes (HTL) and cytotoxic T-lymphocytes (CTL) in vitro as well as in vivo. TLR engagement, however, induces also anti-inflammatory IL-10 secretion from DCs or up-regulation of indoleamine-2,3-dioxygenase (IDO), which renders activated T-cells susceptible to apoptosis. Thus, DCs acquire a continuously changing activation/maturation phenotype: within 24 hours the same DC shifts from the pro- into an anti-inflammatory mode of action.
 
We demonstrated in pre-clinical experiments that limiting LPS/IFN-g-mediated DC maturation to 6 hours enables priming of T-cells in vitro or in vivo, which is superior compared to a maturation of 24-48 hours. This strategy was employed in a randomized clinical efficacy trial for the treatment of glioblastoma multiforme (GBM), the most frequent and most aggressive form of brain cancer, using an individualized DC cancer immune therapy (CIT) concept (GBM-Vax, EudraCT 2009-015979-27, NCT01213407). We randomized 78 patients aged 18-70 years. All patients received first line treatment for GBM, surgery, radiotherapy, and chemotherapy with Temozolomide; in the treatment group, AV0113 was applied as add-on. Primary and secondary objectives were PFS and OS. The study still collects follow up information. The current trend based on approximately two thirds of the patients suggests a strong reduction of deaths in the first year after diagnosis (AV0113 treatment: 18% deaths; control: 45% deaths).
 
Reactions at the AV0113 DC-CIT injection site were mild and included redness and swelling. Some patients developed fever above 40°C that could be therapeutically controlled. The majority of severe adverse events concerned the nervous system but was not different in the two groups (AV0113 treatment: 39%; control: 41%).
 
Randomization for the GBM-Vax study was completed in May 2013; hence we expect a complete data set for the one-year survival in May 2014. If the current trend is confirmed, we expect a first demonstration of efficacy for IL-12-secreting DCs in the treatment of brain cancer.
 

From Nobel Prize to Bedside: Innovative Approaches to Personalized Dendritic Cell Therapies
Charles A. Nicolette, PhD, Argos Therapeutics Inc.

In 2011 Dr. Ralph Steinman was awarded the Nobel Prize in Physiology and Medicine nearly 40 years after his discovery of the dendritic cell. While the importance of the role of dendritic cells in cancer immunology is undisputed, translation of this discovery into efficacious therapies for malignant disease has not yet met with great success. As our understanding of dendritic cell biology continues to evolve, new products are advancing to late-stage clinical development, offering the promise of effective non-toxic interventions that leverage the immune system to control malignant disease. Challenges to the development of dendritic cell-based immunotherapies will be discussed in the context of our experience with AGS-003, a personalized, mRNA-loaded dendritic cell therapy. A Phase 3 pivotal trial was recently initiated with AGS-003 in metastatic renal cell carcinoma patients and, if successful, could be one of the first dendritic cell-based immunotherapies available to patients.
 

Using the Tumor Immunopeptidome to Guide the Design of Personalized DC-based Cancer Vaccines
Gregory Lizee, PhD, MD Anderson Cancer Center

The recent development of clinical-grade monoclonal antibodies specific for T-cell checkpoint blockade molecules like CTLA-4 and PD-1 has ushered in a new era for immunotherapy, and initial clinical trials testing these agents have shown much promise for inducing long-term tumor regressions in a subset of patients. However, despite this encouraging clinical success, the majority of cancer patients still succumb to their disease following an initial response to treatment. A serious current limitation to these immunotherapeutic interventions is a lack of specificity in immune system activation. Dendritic cell-based vaccines, by contrast, have shown great promise for inducing antigen-specific cytotoxic T-cell responses. However, one of the major limitations of this approach is that the vast majority of antigenic targets presented by individual patient tumors are unknown. Having knowledge of these MHC-I ligands will provide unprecedented opportunities to target these tumor targets clinically, resulting in increased effectiveness and less treatment side effects.
 
The specific objective of this research project is to develop an epitope identification method that can reliably and routinely identify MHC-I ligands presented by cancer cells from individual patients. Since recent evidence has shown that the most effective and immunogenic tumor antigens are those containing tumor-specific mutations, we have been specifically searching for those antigens using a combination of next generation sequencing, mass spectrometry-based proteomics, and HLA bioinformatics. We are focusing our initial efforts on melanomas and tumors from lung and colon cancer patients, which accumulate the highest number of somatic mutations. We have now already used our comprehensive antigen identification approach to successfully identify hundreds of non-mutated tumor-associated antigens from individual patient tumor cell lines and surgical biopsies; in addition, we have also identified a number of mutated epitopes, including some that elicit strong autologous T-cell reactivity. Having knowledge of the antigenic peptide targets on patient tumors will allow for the development of more specific and effective immunotherapies, and will greatly broaden the patient base that can be treated with such therapies: most importantly, it will enable us to leave behind the historical limitations such as only being able to target a few known, shared tumor antigens restricted to HLA-A*0201 positive patients. This personalized tumor antigen identification method holds the promise of increasing the efficacy of autologous DC-based cancer vaccines substantially by adding much-needed specificity to current immunotherapeutic approaches.
 

Dendritic Cell Based Immunotherapy of Melanoma: The Brussels' Experience
Kris Thielemans, Vrije Universiteit Brussel

Electroporation of DCs with mRNA encoding the full-length tumor antigens should lead to presentation of many epitopes by the patient’s unique set of HLA molecules. Moreover, electroporation of DC with mRNA also allows the functional modification of the cellular vaccine. To this goal, we provide three different molecular adjuvants to immature, monocyte derived DCs through electroporation with mRNA coding for CD40L, CD70 and caTLR4 or so-called TriMix mRNA.
 
At our institution, clinical trials in pretreated advanced melanoma patients are being performed. These patients are treated with TriMixDC-MEL, a mixture of TriMix-DC co-electroporated with mRNA encoding a fusion of DC.LAMP and 1 of 4 melanoma associated antigens (gp100, tyrosinase, MAGE-C2 or MAGE-A3).
 
Ina pilot clinical trial, 24.10⁶ TriMixDC-MEL cells were administrated solely by the intradermal (ID) route.  Subsequently, a phase IB was conducted to investigate the safety of administrating TriMixDC-MEL by the intravenous (IV) and ID-route.  The ratio of ID/IV administered DC was: Cohort-1: 20.10⁶/4.10⁶ DC [2pts], -2: 12.10⁶/12.10⁶ DC[3pts], -3: 4.10⁶/20.10⁶ [6pts], and -4: 0/24.10⁶ DC [4pts]; DC were administered 4x q2w, and a 5^th administration on w16.  Local skin reactions (gr1-2) were observed in all pts receiving DC ID, flu-like symptoms (< gr2) were observed in 12/21 pts treated ID and in 8/15 pts treated ID/IV.  Post IV infusion chills (gr2) were observed in 3/15 pts.  Inflammatory cytokine release was documented during these chills.  ID administration of TriMixDC-MEL was found to be feasible, safe, effectively stimulating CD8⁺ T-cell responses, but did not result in objective tumor responses.  In contrast, the combined ID/IV administration of TriMixDC-MEL resulted in 2 PR and 2 CR (by RECIST) out of 15 pts (BORR of 27%; ongoing after 24+, 28+, 33+, and 34+ mths).  A confirmed stable disease was documented in four additional patients (for a disease control rate of 53%). From this study we concluded that ID/IV-administration of TriMixDC-MEL as a single-agent cellular immunotherapy is associated with distinct but manageable side-effects and has seemingly superior clinical activity as compared to DC administered solely ID in patients with pretreated advanced melanoma.
 
Ipilimumab (ipi), an anti-CTLA-4 mAb, enhances T-cell function and has established activity in advanced melanoma pts. We aimed to investigate the safety and activity of TriMixDC-MEL combined with ipi. TriMixDC-MEL was administered IV (20.10⁶) and ID (4.10⁶) 1h after ipi infusion (10 mg/kg), q3w for a total of 4 administrations. Maintenance therapy with ipi was allowed q12w for pts free from progression at week 24. The primary endpoint was disease control rate (by irRC). 37 pts initiated treatment.  Local skin injection reactions (gr 1-2) were observed in all pts, flu-like symptoms (gr 1-2) in 20 (54%) pts, post-infusion chills (gr 1-2) in 15 (40%) pts. Immune-related adverse events were observed in 29 (78%) pts [11 (29%) pts had grade 3 or 4 AEs]. Most common were dermatitis (24 pts); hypophysitis/hypopituitarism (6 pts), diarrhea (6 pts), and hepatitis (5 pts). irAEs necessitated systemic corticosteroids in 17 (45%) pts. The best objective tumor response (35 evaluable pts): 5 CR, 5 PR, 9 SD and 16 PD (disease control rate: 54%). Objective responses are currently ongoing in 6/10 pts (11+ -22+ months). This phase II study of TriMixDC-MEL ID/IV in combination with ipi demonstrates anti-melanoma activity in over 50% of the patients with therapy resistant advanced melanoma. Further clinical development of TriMixDC-MEL in combination with immune checkpoint modulators is warranted.
 
Furthermore, TriMixDC-MEL is currently under evaluation in a randomized phase II trial in the adjuvant setting following resection of macrometastases.
 

Collaborative Clinical Studies on Immunotherapies to Treat Ovarian Cancer
Lana E. Kandalaft, PharmD, MTR, PhD

Novel therapeutic strategies are warranted in recurrent ovarian cancer. We report two independent consecutive studies of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy. Thirty one patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from debulking surgery enrolled in two different vaccination studies. First six underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide followed by vaccination with an autologous DC preparation pulsed with freeze-thaw autologous tumor lysate while the other 25 underwent vaccination with an enhanced vaccine of autologous DCs loaded with HOCl-oxidized autologous tumor lysate administered intranodally alone or with bevacizumab or in combination with bevacizumab and cyclophposphamide. Both studies were followed by lymphodepletion and transfer of autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T-cells. Feasibility, safety, biological and clinical efficacy were evaluated.
 
Eleven patients have completed vaccination and T cell transfer to date, while twenty-three additional patients completed vaccination only. Vaccination was well tolerated and elicited tumor-specific T cell responses against various ovarian tumor antigens. Preliminary results demonstrate that patients’ DCs loaded with HOCl-oxidized lysate elicited strong tumor-specific IFN-γ secretions and produced high levels of Th1-priming cytokines and chemokines, including IL-12.
 
Preliminary results indicate a clinical benefit rate 66% in the first study and an incremental clinical benefit increase of 40% in the vaccine only cohort, 60% in the vaccine and bevacizumab cohort and finally 80% cohort 2 and 80% in  the vaccine, bevcacizumab and cyclophosphamide cohort at the end of study. Progression-free survival (PFS) comparing the three cohorts with a matched control group of patients who have undergone secondary debulking and have had cyclophosphamide and bevacizumab but no immunotherapy demonstrates at 500 days from enrollment, PFS of <10% in the control group, 45% in cohort 1, 50% in cohort 2, and 80% in cohort 3 (p=0.0334).
 
Following lymphodepletion, adoptive transfer of vaccine-primed T-cells was well tolerated and resulted in durable reduction of T-regulatory cells and restoration of vaccine-induced antitumor immunity in patients who experienced clinical benefit. One patient exhibited a complete response at end of study and stable disease was observed in 7 out of the 11 patients who completed vaccination and T cell transfer.
 
Our results suggest the use of combinatorial cellular immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of patients with recurrent ovarian cancer is promising yet warrants further investigation.
 

Biomarkers to Indicate Potentially Provenge-Responsive Prostate Cancer Patients
James B. Trager, PhD, Dendreon Corporation

Sipuleucel-T is an autologous cellular immunotherapy approved in the United States and Europe for the treatment of asymptomatic or minimally symptomatic, metastatic castration-resistant prostate cancer (mCRPC).  It is manufactured from peripheral blood mononuclear cells (PBMCs) isolated by leukapheresis. PBMCs are cultured with PA2024, a fusion antigen composed of prostatic acid phosphatase (PAP) fused to granulocyte macrophage-colony stimulating factor (GM-CSF).  A complete course of sipuleucel-T therapy consists of three rounds of product manufacture and infusion at two week intervals.
 
Extensive immunological analyses have been performed in trials evaluating sipuleucel-T in men with mCRPC.  APC activation, antigen-specific immune responses, and cytokine accumulation during product manufacture were studied.  Consistent with an immunological prime-boost model, APC activation, along with antigen-specific IFN-γ ELISpot, proliferative and humoral responses, were greater at the second and third infusions than at the first.  Antigen-specific cellular responses increased after sipuleucel-T treatment.  Following culture with PA2024 we also observed increased CD4+ and CD8+ T cell activation.  The observed T cell activation phenotypes were consistent with the cytokines (IL-2, IL-5, IL-13, and IL-10) produced during manufacture; these cytokines, associated with T cell activation, were elevated at the second and third product manufacture.
 
This talk will provide an overview of sipuleucel-T production, with a focus on product characterization and immune response.  A variety of biomarkers, both baseline and pharmacodynamic, are correlative of clinical response to sipuleucel-T.  We will discuss the biological interpretations of these markers and in particular their implications in understanding the mechanism of action sipuleucel-T.
 

Dendritc Cell Vaccines Targeting Cancer Stem Cells
John S. Yu, MD, Cedars-Sinai Medical Center and Immunocellular Therapeutics, Ltd.

We have developed dendritic cell vaccines for brain cancer. Our group was the first to demonstrate in pre-clinical as well as clinical trials that the dendritic cell vaccine for malignant glioma could be safe and effective in the treatment of brain tumors. Our group was also the first to demonstrate there is a correlation between the immune response and survival using the dendritic vaccine. In a Phase I clinical trial evaluating ICT-107 – a new version of the experimental dendritic vaccine for the treatment of glioblastoma multiforme – showed that patients receiving the vaccine had a greater progression-free survival time than expected in this population. The median progression-free survival time (defined as the time between surgical tumor resection and tumor recurrence) in the 16 newly diagnosed patients enrolled in the trial was 19 months – 12 months longer than the historical progression-free survival time of 6.9 months. At the time the results were reported, six of the 16 patients continued to show no signs of tumor recurrence. The median survival of this group is 38.4 months. In a Phase II, randomized clinical trial, we are studying the safety and efficacy of survival with the vaccine. The therapeutic cancer vaccine has now become a promising area in the treatment of cancer.
 

CVac™ Phase 2 Clinical Study for Treatment of Ovarian Cancer Patients in First & Second Remission (CAN-003 protocol): Manufacturing & Clinical Outcomes
Matthew Lehman, Prima Biomed, Australia

CAN-003 is a randomized, standard-of-care controlled trial of CVac as a maintenance therapy for epithelial ovarian cancer in complete remission after conventional first of second line surgery and chemotherapy. This was the first multicenter and multinational trial with CVac and included two different manufacturing sites for production.
 
Operationally, the trial demonstrated the ability to transfer the manufacturing technology, utilize multicenter autologous cell collections, and it established the comparability of product manufacturing between two sites.
 
Clinically, the CAN-003 trial indicated that CVac is well tolerated and non-toxic. Although there is significant biological variability, CVac induces a broad spectrum mucin 1-specific CD4+ and CD8+ T cell response in this patient population. There is no observed effect on median progression-free survival; however, differing trends were observed between the first and second remission patient groups. Overall survival data is considered too immature for analysis as of September 2013.