Egbert Oosterwijk

Sequence variants at the TERT-CLPTM1L locus associate with many cancer types

The common sequence variants that have recently been associated with cancer risk are particular to a single cancer type or at most two. Following up on our genome-wide scan of basal cell carcinoma, we found that rs401681[C] on chromosome 5p15.33 satisfied our threshold for genome-wide significance (OR = 1.25, P = 3.7 × 10−12). We tested rs401681 for association with 16 additional cancer types in over 30,000 cancer cases and 45,000 controls and found association with lung cancer (OR = 1.15, P = 7.2 × 10−8) and urinary bladder, prostate and cervix cancer (ORs = 1.07−1.31, all P < 4 × 10−4). However, rs401681[C] seems to confer protection against cutaneous melanoma (OR = 0.88, P = 8.0 × 10−4). Notably, most of these cancer types have a strong environmental component to their risk. Investigation of the region led us to rs2736098[A], which showed stronger association with some cancer types. However, neither variant could fully account for the association of the other. rs2736098 corresponds to A305A in the telomerase reverse transcriptase (TERT) protein and rs401681 is in an intron of the CLPTM1L gene.

Immune responses to transgene and retroviral vector in patients treated with ex vivo–engineered T cells

Adoptive transfer of immune effector cells that are gene modified by retroviral transduction to express tumor-specific receptors constitutes an attractive approach to treat cancer. In patients with metastatic renal cell carcinoma, we performed a study with autologous T cells genetically retargeted with a chimeric antibody receptor (CAR) directed toward carbonic anhydrase IX (CAIX), an antigen highly expressed in renal cell carcinoma. In the majority of patients, we observed distinct humoral and/or cellular anti-CAIX-CAR T-cell immune responses in combination with a limited peripheral persistence of transferred CAIX-CAR T cells in the majority of patients. Humoral immune responses were anti-idiotypic in nature and neutralized CAIX-CAR-mediated T-cell function. Cellular anti-CAIX-CAR immune responses were directed to the complementarity-determining and framework regions of the CAR variable domains. In addition, 2 patients developed immunity directed against presumed retroviral vector epitopes. Here, we document the novel feature that therapeutic cells, which were ex vivo engineered by means of transduction with a minimal γ-retroviral vector, do express immunogenic vector-encoded epitopes, which might compromise persistence of these cells. These observations may constitute a critical concern for clinical ex vivo γ-retroviral gene transduction in general and CAR-retargeted T-cell therapy in particular, and underscore the need to attenuate the immunogenicity of both transgene and vector.

Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250

The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)‐associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear‐cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up‐regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor‐associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12‐13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti‐G250 reactivity is rare. Int. J. Cancer 85:865–870, 2000.

Strict regulation of CAIX(G250/MN) by HIF-1alpha in clear cell renal cell carcinoma.

Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel–Lindau (VHL) protein and high, homogeneous expression of the G250MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIXG250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250MN promoter constructs revealed strong promoter activity in G250MN -positive RCC cell lines, but no activity in G250MN -negative cell lines. DNase-I footprint and band-shift analysis demonstrated that Sp1 and HIF-1α proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1α expression and G250MN expression was observed. In contrast, no relationship between HIF-2α expression and G250MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1α and Sp1, in combination with CBP/p300, are crucial elements for G250MN expression in ccRCC, and CAIXG250 can be regarded as a unique HIF-1α target gene in ccRCC.

A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients.

Chimeric monoclonal antibody G250 (WX-G250) binds to a cell surface antigen found on >90% of renal cell carcinoma (RCC). A multicentre phase II study was performed to evaluate the safety and efficacy of WX-G250 in metastatic RCC (mRCC) patients. In all, 36 patients with mRCC were included. WX-G250 was given weekly by intravenous infusion for 12 weeks. Patients with stable disease (SD) or response were eligible to receive additional treatment for 8 weeks. None of the 36 enrolled patients experienced any drug-related grade III or IV toxicity. Only three patients had grade II toxicity possibly related to the study medication. In all, 10 patients had SD and received extended treatment. One complete response and a significant regression was observed during the follow-up of the treatment. Five patients with progressive disease at study entry were stable for more than 6 months after study entry. The median survival after treatment start was 15 months. The weekly schedule of WX-G250 was well tolerated. With a median survival of 15 months after the start of this treatment and two late clinical responses, WX-G250 seems to be able to modulate mRCC. To improve the activity of WX-G250-specific antibody-dependent cellular cytotoxicity and the clinical response rate, currently combinations of WX-G250 with cytokines are in phase II trials.

Immunotherapy for renal cell carcinoma.

Renal cell carcinoma (RCC) is the most prevalent malignancy within the kidney and the incidence is rising. Due to improved radiological evaluation over 50% of the renal cancers are found incidentally. Despite the fact that these incidentalomas are often confined to the kidney, around 50% of all patients diagnosed with kidney cancer will develop systemic disease. Metastatic RCC has a poor prognosis. Traditional treatment modalities like chemo- and radiotherapy show overall response percentages of 2-6%. In view of the observed spontaneous remissions of advanced renal cancer, immune mechanisms have been suggested to play a role in the natural disease course of RCC. At present, several non-specific cytokine regimens are used in the treatment of mRCC, e.g. interleukin-2 and interferon-alpha, in combination or as monotherapy or in combination with substances like 13-cis-retinoic acid and/or 5-fluorouracil. Collective data of trials evaluating cytokine-based therapies for mRCC show an overall response rate of approximately 15%, with 5% of the patients showing complete responses. More importantly, cytokine treatment clearly translates into a significant survival benefit in a subset of patients. Nevertheless, the toxicity profile of these cytokine regimens is significant. With the enhanced knowledge of tumor-immunology, the identification of immunogenic tumor proteins, and antibodies recognizing tumor-associated antigens, new treatment strategies with increased specificity and fewer side effects are of interest. Here we review the different immunotherapeutical modalities currently used as well as new approaches for the treatment of advanced RCC.

Carbonic Anhydrase IX in Renal Cell Carcinoma: Implications for Prognosis, Diagnosis, and Therapy

CONTEXT The clinical management of patients with renal cell carcinoma (RCC) remains difficult, and the development of new diagnostic, prognostic, and therapeutic tools is still required. OBJECTIVE To review the current knowledge on the RCC-associated antigen carbonic anhydrase IX (CAIX) and provide evidence for how this antigen may aid in the clinical management of RCC. EVIDENCE ACQUISITION Clinical papers describing diagnostic, prognostic, and/or therapeutic applications of CAIX in RCC were selected from the Pubmed database. The search was manually augmented by reviewing the reference lists of articles. EVIDENCE SYNTHESIS Expression of CAIX is regulated by the Von Hippel Lindau (VHL) protein (pVHL). Because of the invariable VHL mutational loss in clear-cell RCC (ccRCC) patients, CAIX expression is ubiquitous in ccRCC. Determination of CAIX expression in nephrectomy specimens of RCC patients improves prognostic accuracy; high CAIX expression appears to correlate with a favourable prognosis and a greater likelihood of response to systemic treatment for metastatic disease. Therefore, CAIX expression might be used to stratify metastatic ccRCC (mRCC) patients for systemic treatment. When incorporated into the RCC nomogram, CAIX expression seems to improve diagnostic accuracy for primary RCC as well as mRCC patients, but further evidence is required. Clinical studies with the CAIX-specific monoclonal antibody (mAb) cG250 have provided unequivocal evidence that ccRCC lesions can be imaged with radiolabeled cG250. Results are awaited of a large, randomised trial that aims to establish the value of cG250 imaging for primary RCC. The outcome of another large, placebo-controlled study is awaited to establish the usefulness of CAIX-targeted therapy in the adjuvant setting. Therapeutic trials with high-dose radiolabeled cG250 and CAIX-loaded dendritic cells in mRCC patients are still in phase 1 or 2. CONCLUSIONS CAIX improves diagnostic accuracy and is an attractive target for imaging of and therapy for ccRCC.

Vaccination of patients with metastatic renal cell carcinoma with autologous dendritic cells pulsed with autologous tumor antigens in combination with interleukin-2: a phase 1 study.

Dendritic cells (DC) have been recognized as the most potent antigen presenting cells (APC) of the immune system. We performed a phase 1 study in twelve patients with metastatic renal cell carcinoma (RCC) using autologous immature DC loaded with autologous tumorlysate (TuLy) as a vaccine based on our earlier in vitro observations that such DC can activate tumor-specific cytotoxic T-lymphocytes. The treatment was combined with low-dose interleukin (IL)-2, as this has shown benefit in DC-based therapies. Patients received three intradermal vaccinations at two weekly intervals, and, after each vaccination, IL-2 was administered for 5 consecutive days. In six patients, keyhole-limpet hemocyanin (KLH) was added to the DC culture for immunologic monitoring purposes. In general, DC phenotype was CD14low, CD86high, CD40high, CD80low, and CD83low. We noticed that the number of CD14+ cultured DC increased during treatment. Nevertheless, ovalbumin uptake remained high, underlining that these cells were still functional immature DC. The vaccine was able to elicit cellular anti-KLH responses, emphasizing the ability of the injected DC to mount an immunologic response. However, proliferative responses against TuLy were not detected, and humoral responses against TuLy or KLH were absent. Objective clinical responses were not observed, but extended stable disease was noted. The absence of cellular, humoral, or clinical antitumor responses suggests that the vaccination strategy with immature DC has little benefit for patients with advanced RCC. Nevertheless, this study shows the feasibility of a completely autologous DC and tissue culture methodology for the generation of TuLy pulsed DC.

PD-1 Blockade Augments Th1 and Th17 and Suppresses Th2 Responses in Peripheral Blood From Patients With Prostate and Advanced Melanoma Cancer

Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4 inhibitory receptor (ipilimumab). Enhanced expression of another inhibitory immunoreceptor, programmed death-1 (PD-1), and its ligand, PD-L1, was found to correlate with a poor prognosis in prostate cancer and melanoma. PD-1-blocking antibodies are being developed to modulate antitumor immune responses. To support preclinical and clinical development of anti-PD-1 therapy, we sought to develop biomarker assays that can detect the effect of PD-1-blocking agents in whole blood and peripheral blood mononuclear cells. In this study, we assessed the effect of PD-1 blockade in modulating super antigen (staphylococcus enterotoxin B)-induced and recall antigen (tetanus toxoid)-induced T-cell reactivity in vitro using whole blood and peripheral blood mononuclear cells from patients with advanced melanoma, prostate cancer, and healthy controls. PD-1 blockade was found to shift antigen-induced cellular reactivity toward a proinflammatory Th1/Th17 response, as evidenced by enhanced production of interferon &ggr;, interleukin (IL)-2, tumor necrosis factor &agr;, IL-6, and IL-17 and reduced production of the Th2 cytokines IL-5 and IL-13. It is interesting to note that suppression of Th2 responsivity was seen with whole blood cells only from patients with cancer. Taken together, we identified novel biomarker assays that might be used to determine the functional consequences of PD-1 blockade in peripheral blood cells from patients with cancer. How these assays translate to the local antitumor response remains to be established in a clinical setting.

A Clinical Trial With Chimeric Monoclonal Antibody WX-G250 and Low Dose Interleukin-2 Pulsing Scheme for Advanced Renal Cell Carcinoma

PURPOSE WX-G250 is a chimeric monoclonal antibody that binds to carbonic anhydrase IX(G250/MN), which is present on greater than 95% of RCCs of the clear cell subtype. The suggested working mechanism of WX-G250 is by ADCC. Because the number of activated ADCC effector cells can be increased by a low dose interleukin-2 pulsing schedule, a multicenter study was initiated to investigate whether WX-G250 combined with LD-IL-2 could lead to an improved clinical outcome in patients with progressive RCC. MATERIALS AND METHODS A total of 35 patients with progressive clear cell RCC received weekly infusions of WX-G250 for 11 weeks combined with a daily LD-IL-2 regimen. Patients were monitored longitudinally for ADCC capacity. Radiological assessment of metastatic lesions was performed at week 16 and regularly until disease progression. RESULTS A durable clinical benefit was achieved in 8 of 35 patients (23%), including 3 with a partial response and 5 with stabilization at 24 weeks or greater. Mean survival was 22 months. In general treatment was well tolerated with little toxicity. The number of effector cells increased during treatment but lytic capacity per cell did not increase. ADCC and clinical outcome did not appear to correlate. CONCLUSIONS WX-G250 combined with LD-IL-2 in patients with metastatic RCC is safe and well tolerated. With a substantial clinical benefit and a median survival of 22 months in patients with metastatic RCC who have progressive disease at study entry combination therapy showed increased overall survival compared to WX-G250 monotherapy. Survival was at least similar to that of currently used cytokine regimens but with a favorable toxicity profile.