Drs. Jeong, Rolando and Lerner will discuss the unlabeled use of autologous dendritic cells and a recombinant fusion protein for the treatment of hormone-refractory prostate cancer.

Case

An otherwise healthy 56-year-old African-American male was referred from a primary care provider with several months of increasing urinary hesitancy and sensations of incomplete voiding. Digital rectal exam revealed a nodule at the left base of the prostate. Creatinine and blood urea nitrogen were normal and serum PSA was elevated at 8 ng/mL. Post-voiding residual volume was 220 mL by bladder ultrasound. The patient was referred for transrectal ultrasound of the prostate, which revealed a 120 cc prostate gland without evidence of extra-prostatic extension. (If this nodule were determined to be a prostatic cancer, it would be designated a T2a because it was confined to less than half of one lobe of the prostate and would be preceded by a lower case "c" designating a clinical staging.)

An ultrasonically guided biopsy was performed and histopathologic examination revealed a poorly differentiated adenocarcinoma of the prostate with Gleason 4 + 3 in four of six cores. The Gleason score is used by many pathologists to compensate for the variability of cellular differentiation in different microscopic fields.

CT scanning can detect enlarged lymph nodes but is unreliable for determining penetration of the prostate capsule.

Magnetic resonance imaging (MRI) to determine if the tumor had penetrated the prostate capsule was performed, even though the test is characterized by significant inter-observer variability and relatively poor sensitivity and specificity.

The MRI revealed a large lesion in the left base of the prostate with possible extra-capsular extension. A nuclear medicine bone scan demonstrated no evidence of metastatic lesions. The possible extra-capsular extension and the fact that the patient is African- American suggested that he was at high risk for developing recurrent or metastatic disease.

The patient underwent successful radical retropubic prostatectomy using a unilateral nerve-sparing technique. Pathologic staging revealed a T3aN0 (established extra-capsular extension), Gleason 4 + 3 adenocarcinoma of the prostate with negative surgical margins. Postoperatively, the patient's serum prostate specific antigen (PSA) level was undetectable (<0.1 ng/mL). Following surgery, the patient remained continent with no erectile dysfunction. He presented for planned follow-up to discuss other strategies to maximize his chance of cure.

Over the next two years, the patient remained with no evidence of disease. He had no difficulties with incontinence or erectile dysfunction, and serum PSA levels were undetectable (<0.1 ng/mL). On routine follow-up three years after prostatectomy, the patient's serum PSA level was 1.2 ng/mL. Digital rectal exam revealed an empty prostatic fossa without evidence of recurrent cancer. Two weeks later, the serum PSA level was 2.1 ng/mL and the patient remained asymptomatic. A bone scan and computed tomography (CT) scan of the abdomen and pelvis showed metastatic disease in the T12 vertebral body. Transrectal ultrasonic guided biopsy of his prostatic fossa revealed recurrence of malignancy.

The patient agreed to receive hormonal therapy with the luteinizing hormone-releasing hormone (LHRH) agonist goserelin acetate every three months. With ongoing treatment, the patient's serum PSA returned to undetectable levels (<0.1 ng/mL) and remained undetectable for 10 months after which the serum PSA level kept rising and new multiple bony metastases were seen on radionuclide bone scan. Because the patient's tumor had become refractory to hormone treatment, he received six courses of chemotherapy with docetaxel. The patient developed an episode of febrile neutropenia requiring hospitalization and declined further chemotherapy. He was then offered a vaccine for prostate cancer called Provenge^®. Developed by Dr. Riner Laus, Dendrion Corp., Seattle, WA, in Ronald Levy's National Cooperative Drug Discovery Group (NCDDG), Phase II evaluation of Provenge^® under the direction of Dr. Eric Small at the University of California at San Francisco has been completed.(1)

The Levy-NCDDG approach is to isolate a patient's dendritic cells, pulse them with a peptide derived from prostatic acid phosphatase and re-administer the modified dendritic cells back to the patient. They are proceeding to a randomized Phase III trial based on a notable decrease in disease progression in Phase I and II trials.

The patient agreed to take part in a clinical trial of Provenge^® dendritic cell immunotherapy. Peripheral blood mononuclear cells were harvested at weeks 0, 2, 4 by standard 2-blood volume leukapheresis. [Leukapheresis is a technique to remove leukocytes from a patient's circulation using a continuous flow centrifuge through which the patient's blood is circulated. The volume of blood circulated was twice the patient's whole blood volume in this case.] Mobilization with a colony-stimulating factor was not required. The leukapheresis products were prepared at a local blood bank and transported within one hour to the regional cell processing facility.

Two sequential buoyant density centrifugation steps collected dendritic cell precursors. The dendritic cell precursors were washed and incubated in media with the appropriate target antigen (PA2024). PA2024, the target antigen, is a fusion protein consisting of full-length human prostatic acid phosphatase (PAP) and full-length human granulocyte-macrophage colony-stimulating factor (GM-CSF). After incubation for two days, the cells were washed and formulated at the CD54 + dendritic cell dose of 200 x 10⁶ cells in 250 mL of lactated Ringers' solution.

Two days after the last leukapheresis, the patient received an infusion of Provenge^®. He was treated with Provenge^® on weeks 0, 4, 8 and 24. He had a mild febrile infusion reaction and mild myalgias treated with acetaminophen and diphenhydramine at week 4 and subsequently used as prophylaxis. After the first infusion, he developed transient mild discomfort in the prostate area and an elevated PSA that decreased back to baseline in two weeks. It was thought to be mild prostatitis secondary to Provenge^®. His pretreatment T cells did not secrete either the cytokines interferon-gamma (IFN-gamma) or interleukin-4 (IL-4), which are markers of T-cell activation in response to PA2024.

However, T cells collected after treatment with Provenge^® secreted IFN-r but not IL-4 in response to PA2024, indicating a helper T type 1 response. In addition, assays of cytokine secretion by single lymphocytes showed that the frequency of cells secreting IFN-r in response to PA2024 increased from undetectable (<1/10⁶ cells) to 1/5000 cells. At 30 weeks, his PSA level declined more than 50% and the patient had a significant decrease in evidence of metastases on his radionuclide bone scan.

Dendritic Cell Immunotherapy for Malignancy

Dendritic cells (DC) are either stromal- or bone marrow-derived. In common usage, the term dendritic cell usually refers to bone marrow-derived cells that are found in the blood or in tissues. DCs are the most potent antigen presenting cells (APCs) and the only APCs that can prime an immune response by T cells that have not previously been exposed to antigen.

They are the single most central player in all immune responses -- both innate and adaptive. DCs are exceptionally strong potentiators of the immune response in inflammatory conditions, yet are also critical to the induction and maintenance of self-tolerance in the steady state.

DCs are normally found in the steady state in peripheral tissues and/or the circulation. Semi-mature DCs continuously present self-antigens, and probably harmless non-self-antigens, in secondary lymphoid organs to maintain peripheral tolerance and anergy. Harmful pathogens or other dangerous insults to the steady state can lead to inflammation, with full maturation and activation of each DC subset. Physiologically, this stimulus is usually provided by a microbial pathogen or product, or by activated CD4+ T cells expressing CD40L.

In vitro, there are at least four types of DCs defined under cytokine-driven conditions. These include CD14+ blood monocyte-derived DCs, dermal DCs or interstitial DCs, Langerhans cells and plasmacytoid DCs.

The cytokine-driven DC progeny, however, would not be found in vivo except under inflammatory conditions. Blood monocyte-derived DCs are the most accessible, biologically flexible precursors and first responders to pathogens, allergens and other causes of inflammation.

DCs enhance the reactivity of resting natural killer cells (NK), which otherwise respond to the aggregate of activating and inhibitory signals on their targets. Among conventional DCs, blood monocyte-derived DCs are the most potent stimulators of NK cell proliferation and cytotoxicity. Langerhans cells localize to epithelial surfaces of skin and mucosa and dermal DCs localize to the sub-epithelial tissues of the dermis in skin and the interstitium of solid organs.

APCs (antigen presenting cells) have major histocompatibility complex (MHC) molecules of class I and II on their surface. DCs share most of these features. Antigens acquired from the extracellular environment are typically processed onto class II MHC molecules, whereas class I MHC molecules bear antigens synthesized in the cytosolic compartment. DCs also have the capacity to present exogenous antigens on class I MHC complexes.

But DCs alone are not enough to induce an optimal T-cell response. The optimal generation of T-cell responses needs several stimulatory signals. The first signal is provided by activation of the T-cell receptor, with the antigen associated with the appropriate MHC molecule. The second, or co-stimulatory, signal results from interactions between several co-receptors on T cells and counter receptors on antigen-presenting cells. Stimulation of the T-cell receptor in the absence of co-stimulatory signals induces T-cell anergy and peripheral tolerance. Thus, effective priming of an adequate anti-tumor T-cell response needs presentation of tumor-associated antigens with appropriate stimulatory molecules.

Most tumor antigens are poor immunogens because they are self-antigens, or self-differentiation antigens, to which there is considerable tolerance. DCs provide a potential solution to this challenge by coupling tumor antigens with all of the requisite co-stimulatory ligands, cytokines and chemokine-directed migration to secondary lymphoid organs. In those lymphoid areas, DCs can stimulate incoming T cells to exit via efferent lymph into the periphery as cytolytic and helper T cell effectors. Most tumor-associated antigens used so far in the clinical setting (e.g., carcinoembryonic antigen [CEA]) are not vital to the metabolism of malignant cells. Accordingly, down-regulation of these cells with non-vital antigens will have no significant role in tumor metabolism.

Antigens that are needed by cancer cells to survive are a new class of tumor-associated antigens, e.g., the anti-apoptotic protein survivin, which have been investigated in preclinical and clinical studies. A further development of this strategy is to target tumor cells indirectly by immunization of the host against antigens selectively expressed by tumor-associated endothelial cells, thus inducing tumor necrosis by blocking the blood supply.

This targeting occurs through up-regulated vascular-endothelial growth factor receptor 2 (FLK-1) of proliferating endothelial cells in the tumor vasculature.

Designing a DC Vaccine

Challenges to designing the optimal DC vaccine include the choice of DC and antigen subsets. Other unknowns include optimal antigen loading strategies such as peptide pulsing, overlapping polypeptide pulsing, cross-presentation of dying tumor cells, fused tumor-DC heterokaryons, DNA or RNA transfer with or without a vector construct, frequency and route of immunization, and cell dose. Major challenges remain in terms of harnessing the capacity of DCs for simultaneous presentation of multiple tumor antigens tailored to their own MHC molecules, rather than presentation of only a few peptides with defined MHC restrictions. Migration of DCs to draining lymph nodes also requires optimization after vaccination.

Preclinical studies aimed at eliciting prostate-specific immunity demonstrated that dendritic cells loaded with an engineered antigen-cytokine fusion protein (PA2024) consisting of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce strong cellular immune responses in vivo to tissues and tumors that express PAP.

Dendritic cells pulsed only with PAP elicited significantly weaker immune responses, indicating an important role for the GM-CSF portion of the fusion protein in antigen presentation. Also, protein-pulsed dendritic cells may be more effective than single peptide-pulsed dendritic cells for stimulating immunity because of the larger repertoire of antigens present in the protein and the resulting ability to elicit both CD4+ helper cells and CD8+ effector cells.

Provenge^® Clinical Trial

Dendritic cells are essential for eliciting cellular immunity in this model, as injections of the PAP-GM-CSF fusion protein alone and injections of PAP in Freund's adjuvant elicited antibody responses but not cellular immune responses to PAP. Based on these preclinical observations, a dendritic-cell product (Provenge^®; Dendreon Corp, Seattle, WA), consisting of autologous dendritic cells loaded with the human PAP-GM-CSF fusion protein, was developed. Clinical testing of Provenge^® was undertaken in hormone refractory prostate cancer patients in a phase I/II trial.

Overall the treatment was well tolerated. There was no treatment-related hematologic, hepatic or renal toxicity. The adverse events were febrile reactions, mild myalgias, mild fatigue and mild urinary complaints, including obstructive voiding symptoms, incontinence, urgency and nocturia. There was no evidence for the development of an autoimmune disease caused by cross-reactivity between the PAP antigen and a normal tissue component. This lack of cross-reactivity with normal tissue antigens was predicted from the lack of PAP expression by normal tissues other than the prostate.(8) An immune response to PAP expressed by normal prostate tissue could result in prostatitis. Although five men developed urinary symptoms, none of these were clearly caused by treatment-induced prostatitis.(1)

Immunization resulted in proliferation of Th1 cells. T cells can be separated into two distinct groups based on the type of cytokines the cells secrete. Th1 cells secrete IFN-gamma whereas Th2 cells secrete IL-4 and IL-10. The patients' pretreatment T cells did not secrete either IFN-gamma or IL-4 in response to PA2024. However, T cells collected after treatment with Provenge^® secreted IFN-gamma but not IL-4 in response to PA2024, indicating a Th1-type response thought to be important for host immunity to tumors. In addition, assays of cytokine secretion by single lymphocytes showed that the frequency of cells secreting IFN-gamma in response to PA2024 increased.(1)

Out of a total of 31 patients, three patients had a more than 50% decrease in serum PSA, and three patients had 25% to 49% decreases in PSA. No improvements in bone scans or soft tissue disease were observed. The median time to disease progression for the patients in the phase I trial was 12 weeks, and the median time to progression for the phase II patients was 29 weeks. Seven of the 19 phase II patients had not progressed by the end of the planned 1-year follow-up period. The median time to disease progression was 34 weeks for patients who developed an immune response (N=20) compared with 13 weeks for patients who did not (N=11) (P<.027). The median time to disease progression was 31.7 weeks for patients who received more than 100 x 10⁶ cells/infusion compared with 12.1 weeks for patients who received fewer cells. The difference between the two groups was statistically significant (P=.013).(1)

In Progress

Currently, a randomized placebo-controlled phase III trial of Provenge^® dendritic cell vaccination for treatment of patients with advanced prostate carcinoma is ongoing. Other companies, such as Northwest Biotherapeutics and IDM Biotech, are also conducting trials of dendritic cell vaccine therapy. Immunotherapy not based on dendritic cells is also being developed, such as antibodies directed against tumor antigens.