Michael Erdmann

Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection

Dendritic cell (DC) vaccination approaches are advancing fast into the clinic. The major obstacle for further improvement is the current lack of a simple functionally “closed” system to generate standardized monocyte-derived (mo) DC vaccines. Here, we significantly optimized the use of the Elutra counterflow elutriation system to enrich monocytic DC precursors by (1) developing an algorithm to avoid red blood cell debulking and associated monocyte loss before elutriation, and (2) by elutriation directly in culture medium rather than phosphate-buffered saline. Upon elutriation the bags containing the collected monocytes are simply transferred into the incubator to generate DC progeny as the final “open” washing step is no longer required. Elutriation resulted in significantly more (≥2-fold) and purer DC than the standard gradient centrifugation/adherence-based monocyte enrichment, whereas morphology, maturation markers, viability, migratory capacity, and T cell stimulatory capacity were identical. Subsequently, we compared RNA transfection, as this is an increasingly used approach to load DC with antigen. Elutra-derived and adherence-derived DC could be electroporated with similar, high efficiency (on average >85% green fluorescence protein positive), and appeared also equal in antigen expression kinetics. Both Elutra-derived and adherence-derived DC, when loaded with the MelanA peptide or electroporated with MelanA RNA, showed a high T cell stimulation capacity, that is, priming of MelanA-specific CD8+ T cells. Our optimized Elutra-based procedure is straightforward, clearly superior to the standard gradient centrifugation/plastic adherence protocol, and now allows the generation of large numbers of peptide-loaded or RNA-transfected DC in a functionally closed system.

Denileukin diftitox (ONTAK) induces a tolerogenic phenotype in dendritic cells and stimulates survival of resting Treg

Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.

Twelve-year survival and immune correlates in dendritic cell–vaccinated melanoma patients

BACKGROUND Reports on long-term (≥10 years) effects of cancer vaccines are missing. Therefore, in 2002, we initiated a phase I/II trial in cutaneous melanoma patients to further explore the immunogenicity of our DC vaccine and to establish its long-term toxicity and clinical benefit after a planned 10-year followup. METHODS Monocyte-derived DCs matured by TNFα, IL-1β, IL-6, and PGE2 and then loaded with 4 HLA class I and 6 class II-restricted tumor peptides were injected intradermally in high doses over 2 years. We performed serial immunomonitoring in all 53 evaluable patients. RESULTS Vaccine-specific immune responses including high-affinity, IFNγ-producing CD4+ and lytic polyfunctional CD8+ T cells were de novo induced or boosted in most patients. Exposure of mature DCs to trimeric soluble CD40 ligand, unexpectedly, did not further enhance such immune responses, while keyhole limpet hemocyanin (KLH) pulsing to provide unspecific CD4+ help promoted CD8+ T cell responses - notably, their longevity. An unexpected 19% of nonresectable metastatic melanoma patients are still alive after 11 years, a survival rate similar to that observed in ipilimumab-treated patients and achieved without any major (>grade 2) toxicity. Survival correlated significantly with the development of intense vaccine injection site reactions, and with blood eosinophilia after the first series of vaccinations, suggesting that prolonged survival was a consequence of DC vaccination. CONCLUSIONS Long-term survival in advanced melanoma patients undergoing DC vaccination is similar to ipilimumab-treated patients and occurs upon induction of tumor-specific T cells, blood eosinophilia, and strong vaccine injection site reactions occurring after the initial vaccinations. TRIAL REGISTRATION ClinicalTrials.gov NCT00053391. FUNDING European Community, Sixth Framework Programme (Cancerimmunotherapy LSHC-CT-2006-518234; DC-THERA LSHB-CT-2004-512074), and German Research Foundation (CRC 643, C1, Z2).

Erosive pustular dermatosis of the leg in a patient with ankylosing spondylitis: neutrophilic dysfunction as a common etiological factor?

A 73-year-old Caucasian man presented with a 6-week history of erosive lesions of both lower legs, which showed no improvement upon topical Betametason treatment. Additionally, he suffered from a chronic venous insufficiency, ankylosing spondylitis for over 12 years, hypothyroidism, and anemia. Medication consisted of prednisolone 5 mg, levothyroxine 75 μ g daily, and iron substitution. Examination showed superficial erosions with crusting on both shins measuring approximately 7 × 12 cm. Removal of the crusting revealed multiple pinhead-sized, nonfollicular pustules coalescing into pustular lakes strictly limited to circumscribed painless, erythematous, moist erosions on both lower legs (Fig. 1, left). Light microscopy revealed parakeratosis and subcorneal pustules. The papillary dermis showed a diffuse lymphocytic infiltrate and numerous granulocytes migrating into the epidermis (Fig. 2). Direct immunofluorescence demonstrated discontinuous granular C3 deposits at the basal membrane and minor dermal vessel associated IgM deposits. Indirect immunofluorescence was negative. Microbiology revealed sterile pustules. Laboratory examinations disclosed an elevated C-reactive protein 112 mg/l (< 5 mg/l), borderline serum IgG and IgA levels, and an inflammatory-associated anemia: hemoglobin 9.5 g/dl (> 13 g/dl); 3.76 × 10 6 erythrocytes per μ l (> 4.2 × 10 6 / μ l); ferritin 22 μ g/ml (> 160 μ g/ml).

Preclinical evaluation of NF-κB-triggered dendritic cells expressing the viral oncogenic driver of Merkel cell carcinoma for therapeutic vaccination

Background: Merkel cell carcinoma (MCC) is a rare but very aggressive skin tumor that develops after integration of a truncated form of the large T-antigen (truncLT) of the Merkel cell polyomavirus (MCV) into the host’s genome. Therapeutic vaccination with dendritic cells (DCs) loaded with tumor antigens is an active form of immunotherapy, which intends to direct the immune system towards tumors which express the respective vaccination antigens. Methods: Cytokine-matured monocyte-derived DCs of healthy donors and MCC patients were electroporated with mRNA encoding the truncLT. To permit major histocompatibility complex (MHC) class II next to class I presentation, we used an RNA construct in which the antigen was fused to a DCLamp sequence in addition to the unmodified antigen. To further improve their immunogenicity, the DCs were additionally activated by co-transfection with the constitutively active nuclear factor (NF)-κB activator caIKK. These DCs were used to stimulate autologous CD8+ T-cells or a mixture of CD4+ and CD8+ T-cells. Then the percentage of T-cells, specific for the truncLT, was quantified by interferon (IFN)γ ELISpot assays. Results: Both the truncLT and its DCLamp-fusion were detected within the DCs by flow cytometry, albeit the latter required blocking of the proteasome. The transfection with caIKK upregulated maturation markers and induced cytokine production. After 2–3 rounds of stimulation, the T-cells from 11 out of 13 healthy donors recognized the antigen. DCs without caIKK appeared in comparison less potent in inducing such responses. When using cells derived from MCC patients, we could induce responses for 3 out of 5 patients; however, here the caIKK-transfected DCs did not display their superiority. Conclusion: These results show that optimized DCs are able to induce MCV-antigen-specific T-cell responses. Therapeutic vaccination with such transfected DCs could direct the immune system against MCC.

Real world experience in low-dose ipilimumab in combination with PD-1 blockade in advanced melanoma patients

Dual immune-checkpoint blockade with the anti-PD-1 antibody nivolumab (1 mg/kg) and standard-dose ipilimumab (3 mg/kg) is the mainstay of immunotherapy in advanced melanoma and it is approved since 2016. However, severe side effects (grade 3/4) occur in up to 60% of the patients. Recently, clinical trials have shown similar anti-tumor activity with a more favorable toxicity profile in patients treated with low-dose ipilimumab (1 mg/kg) and standard-dose pembrolizumab (2 mg/kg). In this study we report on the real-world experience of this dosing regime in advanced melanoma patients not eligible for clinical trials. A total of 33 patients with metastatic melanoma (24 with cutaneous and 9 with uveal melanoma) were assessed, retrospectively. Brain metastases were present in 33% of the patients and lactate dehydrogenase was elevated in 70%. Overall response rates were 38% and 0% in cutaneous melanoma and uveal melanoma respectively. Median overall survival was not reached in cutaneous melanoma and was 18 months in uveal melanoma. In 18% of the patients at least one treatment-related severe adverse event was observed. Our observation that the combination of standard dose pembrolizumab and low-dose ipilimumab has a favorable toxicity profile yet anti-tumor activity comparable to the approved standard-dose combination regime in advanced patients not suitable for enrollment in clinical trials is encouraging.

Automated closed-system manufacturing of human monocyte-derived dendritic cells for cancer immunotherapy

Dendritic cell (DC)-based vaccines have been successfully used for immunotherapy of cancer and infections. A major obstacle is the need for high-level class A cleanroom cGMP facilities for DC generation. The CliniMACS Prodigy® (Prodigy) represents a new platform integrating all GMP-compliant manufacturing steps in a closed system for automated production of various cellular products, notably T cells, NK cells and CD34+ cells. We now systematically tested its suitability for producing human mature monocyte-derived DCs (Mo-DCs), and optimized it by directly comparing the Prodigy approach to our established standard production of Mo-DCs from elutriated monocytes in dishes or bags. Upon step-by-step identification of an optimal cell concentration for the Prodigys CentriCult culture chamber, the total yield (% of input CD14+ monocytes), phenotype, and functionality of mature Mo-DCs were equivalent to those generated by the standard protocol. Technicians labor time was comparable for both methods, but the Prodigy approach significantly reduced hands-on time and high-level clean room resources. In summary, using our optimized conditions for the CliniMACS Prodigy, human Mo-DCs for clinical application can be generated almost automatically in a fully closed system. A significant drawback of the Prodigy approach was, however, that due to the limited size of the CentriCult culture chamber, in contrast to our standard semi-closed elutriation approach, only one fourth of an apheresis could be processed at once.

Senescence markers - predictive for response to checkpoint inhibitors?: Senescence markers - predictive for response to checkpoint inhibitors?

Recent studies suggest that the age‐related remodeling of the immune system, known as immunosenescence, could impact the efficacy of immune checkpoint inhibitors in leukemia or nonsmall cell lung cancer. We investigated whether senescence markers can predict response to checkpoint inhibitor therapy in melanoma patients. The peripheral blood of patients with newly diagnosed, untreated metastatic melanoma was analyzed by flow cytometry to correlate the frequency of senescence markers with clinical response as measured by RECIST after 12 weeks of treatment with immune checkpoint inhibitors. The loss of surface markers CD27 and CD28 or the expression of Tim‐3 and CD57 on T cells was associated with resistance to checkpoint inhibitor blockade, presenting these phenotypes as possible predictive biomarkers for checkpoint inhibitor therapy. Immunosenescence seems to impact on the response to checkpoint inhibitor therapy in melanoma patients. Thus, lymphocyte phenotyping for senescence markers, with the introduction of immunosenescence panels, could be predictive for checkpoint inhibitor response.

Rapid development of cutaneous melanoma metastases after herpes zoster infection in a radiotherapy field

Radiotherapy is used successfully for locally advanced primary or metastatic melanoma, and for adjuvant or palliative care. However, as it causes both acute and chronic skin damage, this results in an increased risk of skin cancer, particularly of nonmelanoma types. Additionally, the risk of herpes zoster (HZ) may also be increased in the radiation area. We report a patient with stage III metastatic melanoma (by American Joint Committee on Cancer staging 2009) who rapidly developed cutaneous metastases following HZ infection in a radiation area only 4 months after completing adjuvant radiotherapy. A 50-year-old man presented with an ulcerated nodular melanoma on his back, of Breslow thickness 6.4 mm (BRAF wild-type and NRAS-mutated). Wide excision and sentinel lymph node (SLN) biopsies in both axillae were performed, and the SLNs were positive. Radical lymph node dissection was carried out in the right axillary region (removal of 2 of 17 affected lymph nodes) and the left axillary region (removal of 2 of 17 affected lymph nodes) demonstrated a metastatic lymph node in the right axillary region with extranodal growth. Thus, adjuvant local radiation therapy (55.8 Gy in 31 fractions) was applied to the right axillary lymph nodes, including the lateral infraclavicular region and the patient’s back, and adjuvant immunotherapy with interferon (IFN)-alfa was initiated. Four months after completing radiation therapy, the patient presented with painful blistering along the right thoracic dermatomes C8 to Th1, including the radiotherapy field, with associated paraesthesia. Given the typical clinical presentation and the patient’s symptoms, HZ infection was diagnosed and successfully treated with aciclovir 10 mg/kg body weight intravenously three times daily for 5 days (Fig. 1a). Six days later, the patient re-presented, this time with black nodules on his right upper chest, which measured 2–5 mm in diameter (Fig. 1b). Additional black nodules developed rapidly over the next 2 weeks (Fig. 1c,d). Interestingly, the nodules were primarily located within the previously irradiated thoracic dermatomes C8 to Th1. Histopathology confirmed a centrally necrotic dermal metastasis with multiple mitoses (Fig. 2). Computed tomography scans of the neck, trunk and abdomen, as well as MRI of the brain, ruled out distant metastases. IFN-alfa was stopped, and systemic treatment with anti-PD1 antibody was initiated. There are only a few cases in the literature on melanoma development in radiation areas or after HZ infection. Trefzer et al. reported on two patients with primary melanoma or melanoma metastases in radiation fields 6 and 43 years after radiotherapy, respectively. However, unlike in our patient, neither patient had a history of prior HZ infection. Zalaudek et al. presented a case of zosteriform cutaneous melanoma metastases arising in a dermatome previously infected by HZ. Recurrent melanoma presenting with zosteriform metastases was also reported by North et al.; their patient was initially treated for HZ, and 3 weeks later, melanoma skin metastasis was diagnosed and appropriate treatment was initiated. They proposed that their patient actually did not have HZ infection, but that the skin metastases presented in a zosteriform pattern due to lymphatic spread from a previously excised paravertebral skin metastasis. In both cases, however, the accurate pathophysiological correlation for a zosteriform distribution of metastases remained unclear. Correspondence: Dr Med Ugur Uslu, Department of Dermatology, FriedrichAlexander-University Erlangen-N€ urnberg (FAU), Universit€atsklinikum Erlangen, Ulmenweg 18, D-91054 Erlangen, Germany E-mail: ugur.uslu@uk-erlangen.de

Intracorneal Hematoma Showing Clinical and Dermoscopic Features of Acral Lentiginous Melanoma

Intra- and subcorneal hematoma, a skin alteration seen palmar and plantar after trauma or physical exercise, can be challenging to distinguish from in situ or invasive acral lentiginous melanoma. Thus, careful examination including dermoscopic and histologic assessment may be necessary to make the correct diagnosis. We here present a case of a 67-year-old healthy female patient who presented with a pigmented plantar skin alteration. Differential diagnoses included benign skin lesions, for example, hematoma or melanocytic nevus, and also acral lentiginous melanoma or melanoma in situ. Since clinical and dermoscopic examinations did not rule out a malignant skin lesion, surgical excision was performed and confirmed an intracorneal hematoma. In summary, without adequate physical trigger, it may be clinically and dermoscopically challenging to make the correct diagnosis in pigmented palmar and plantar skin alterations. Thus, biopsy or surgical excision of the skin alteration may be necessary to rule out melanoma.