bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combination immunotherapy with rituximab and local delivery of CpG oligonucleotide in a syngeneic murine model expressing human CD20
Authors: Hurvitz S. A. , Betting D. , Kafi-Tehrani K. , Golay J. , Olafsen T. , Wu A. M. , Timmerman J. M. 
Published: March 2020

2502 Background: Immunostimulatory agents such as CpG oligonucleotides (CpG) may improve the efficacy of rituximab (R) by augmenting antibody-dependent cellular cytotoxicity. We have generated a murine B cell lymphoma expressing human CD20 (38C13-huCD20) for growth in syngeneic wild-type (WT) mice to efficiently study combination immunotherapy in an immunocompetent model. Methods: Groups of mice were inoculated s.c. with tumor cells and after tumor establishment, were treated with R on days 1 and 3, and either intratumoral (i.t.) or systemic (intraperitoneal, i.p.) CpG days 1, 2, 3, 5, 7 and 9. Tumor size was monitored daily and animals sacrificed when tumors reached 1.4 cm. Results: 38C13-huCD20 tumors were shown to express high levels of CD20 by flow cytometry, immunohistochemistry and microPET. While single agent R prevented growth of 100% of tumors when given on the day of inoculation, therapy of established tumors with either R or CpG alone provided no cures. In contrast, R plus i.t. CpG resulted in complete regressions (CR) of tumors in some mice. The route of CpG delivery was critical as only i.t., not systemic (i.p.) CpG combined with R led to CRs. All mice with CR survived challenge with a lethal dose of WT tumor cells, suggesting induction of immunity against non-human CD20 tumor antigens. Cyclophosphamide (Cy) was used to cytoreduce bulky tumors prior to treatment with R/CpG. Cy alone resulted in initial tumor shrinkage but no cures. In this stringent setting, there were no survivors after Cy/R. Long term tumor-free survivors were seen only in mice receiving i.t. CpG ± R. Addition of i.t. CpG significantly improved survival over Cy/R (p=0.018), while addition of i.p. CpG did not (p=0.24). Adding a second cycle of R/CpG i.t. on day 14 after Cy further improved survival (day 55) compared to 2 cycles of R alone (p=0.01) or 2 cycles of i.t. CpG alone (p=0.002). Conclusions: We have identified a novel combination immunotherapy resulting in eradication of an aggressive B cell lymphoma. Admininstration of CpG into the tumor bed was critical in enhancing the R effect. Ongoing studies are evaluating underlying immunologic mechanisms. Such models should allow identification of promising strategies appropriate for translation into clinical trials. [Table: see text]
Interleukin-21 (IL-21): Tolerability and anti-tumor activity following two 5-day cycles in patients with stage IV melanoma (MM) or renal cell carcinoma (RCC)
Authors: Thompson J. A. , Curti B. D. , Redman B. G. , Weber J. S. , Agarwala S. S. , Sievers E. L. 
Published: March 2020

2505 Background: IL-21 enhances NK cell activity and the proliferation and activation of CD8+ T cells. A Phase 1 outpatient dose-escalation trial of IL-21 in patients with MM or RCC established a maximum tolerated dose and demonstrated biologic effects as measured by increased soluble CD25 levels and decreased lymphocyte counts. Partial responses (PR) were observed in 2 patients with RCC (J Immunother. 2005; 28:641–642). Methods: IL-21 was administered by intravenous push at 30 μg/kg/day for two 5-day cycles separated by a 9- or 16-day rest to a total of 34 patients (18 MM and 16 RCC) to further characterize safety and evaluate preliminary signs of anti-tumor activity. Results: All patients received 2 cycles of treatment with the exception of one patient who discontinued secondary to pleural effusion. Common drug-related Grade 1–2 adverse events (AEs) included pyrexia (n=27), fatigue (n=25), chills (n=18), headache (n=18), and rash (n=16). Related Grade 3 AEs included pleural effusion, abdominal pain, hypophosphatemia, and thrombocytopenia (n=1 each). Laboratory abnormalities commonly observed were decreased lymphocytes (n=31), platelets (n=31), albumin (n=29), and hemoglobin (n=27); increased triglycerides (n=27) and ALT (n=26). Except for lymphopenia (an expected pharmacodynamic response), most toxicities were Grade 1–2. AEs and laboratory abnormalities were generally transient. Final study results, including response data, will be presented. To date, of 28 patients evaluable for response (i.e., those who received at least 8 of 10 doses and have had a final evaluation), one PR has been confirmed in a patient with MM and 18 patients have stable disease two months following initiation of therapy. Conclusions: Two 5-day outpatient cycles of IL-21 at 30 μg/kg/day have been reasonably well tolerated and have demonstrated preliminary evidence of anti-tumor activity in patients with advanced MM or RCC. [Table: see text]
A phase II trial of denileukin diftitox to treat refractory advanced-stage ovarian cancer
Authors: Barnett B. G. , Ruter J. , Brumlik M. J. , Kryczek I. , Cheng P. J. , Zou W. , Curiel T. J. 
Published: March 2020

2506 Background: Denileukin diftitox is a fusion toxin consisting of interleukin-2 genetically fused to diphtheria toxin. It is approved to treat cutaneous T cell lymphoma. Our recent Phase I trial demonstrated that denileukin diftitox depletes human regulatory T cells (Tregs), and is associated with improved measures of T cell immunity and clinical improvements in ovarian cancer. This Phase II trial tests whether denileukin diftitox has clinical activity in the treatment of relapsed epithelial ovarian cancer. Methods: Patients with Stage III or IV ovarian cancer who have failed first-line therapy with optimal surgical debulking and platinum-based chemotherapy were eligible. Denileukin diftitox was given at 12 mcg/kg once monthly. In the Phase II design, patients received no chemotherapy for at least two weeks and no immune or radiation therapy for at least four weeks prior to enrollment. Infusion pretreatment includes antihistamines, acetaminophen and anti-emetics, but no corticosteroid. Results: Six patients (F1,2,4–7) have been treated to date. F1 experienced CA-125 stabilization and approximately 20% reduction in her pelvic mass after two cycles, but was then withdrawn for hypoalbuminemia. F2 experienced CA-125 stabilization after two cycles and remains stable through four cycles. F4 experienced a 20% drop in CA-125 after two cycles, but then progressed. F5 progressed after one cycle. Treatment was recently initiated for F6–7. Existing immune data to date confirm that denileukin diftitox treatment in the Phase II trial is associated with reductions in phenotypic CD4+CD25hi blood Tregs and increased CD3+IFN-γ+ T cells consistent with our Phase I results. Studies of Treg function, T cell cytokine production and CD4+CD25+ T cell FOXP3 content using saved clinical material will be undertaken when full laboratory operations resume. The trial continues to accrue patients. Conclusions: These data suggest that denileukin diftitox depletes Tregs in ovarian cancer. The link between Treg depletion and any observed immune changes or clinical effects is under active investigation, and remains to be determined. No significant financial relationships to disclose.
Impaired Ca++ mobilization in rituximab-resistant cells (RRCL) is associated with changes in the structure of CD20 antigen, down-regulation of Bax/Bak pro-apoptotic proteins and up-regulation of the endoplasmic reticulum (ER) Ca++ pump protein SERCA-3
Authors: Hernandez-Ilizaliturri F. J. , Kaur H. , Bhinder A. , Olejniczak S. , Knight J. , Czuczman M. S. 
Published: March 2020

2516 Ca++ mobilization leading to apoptosis has been observed following rituximab biding to CD20 in lymphoma cells. Extending rituximab (R) exposure (e.g. as via R maintenance regimens) could potentially accelerate the emergence of resistance to rituximab. To define the molecular basis for rituximab resistance we developed several RRCL and demonstrated changes in CD20 expression/structure and membrane reorganization following rituximab therapy. Specifically, changes in the N-terminal and the C-terminal region of the internal domain of CD20 were observed in RRCL as determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. In our current work we evaluated the effects that CD20 structure/expression changes have upon Ca++ mobilization. Extracellular and intracellular Ca++ mobilization was measured by flow cytometric analysis using FLUO-3 AM (acetoxymethyl ester). Optimization of the Ca++ indicator and calibration curves were performed for each cell line. Raji and RRCL were labeled under optimal conditions with FLUO-3 AM/Pluronic Acid F-127. Subsequently, cells were then re-suspended in HANKS media with or without Ca++ and exposed to rituximab or isotype (10μg/ml) ± human serum (25%). Surface CD20 expression was similar between Raji cells and RRCL. Expression of pro- or anti-apoptotic proteins and Ca++ regulatory proteins SERCA3, SERCA2 and Calreticulin was also determined by western blotting. In vitro exposure of Raji cells to rituximab + HS resulted in Ca++ mobilization even in Ca++ depleted media. Notably, Ca++ mobilization was impaired in RRCL when compared to Raji parental cells. In addition, down-regulation of Bax/Bak and up-regulation of SERCA3 was demonstrated in RRCL. Our data suggest that the acquirement of rituximab resistance is associated with changes in the intracellular domain of CD20 and in Ca++ regulator/pro-apoptotic proteins (SERCA3 and Bax/Bak) resulting in a decrease in the intracellular mobilization of Ca++. No significant financial relationships to disclose.
Tumor-specific peptide vaccination in newly-diagnosed patients with GBM
Authors: Heimberger A. B. , Hussain S. F. , Aldape K. , Sawaya R. , Archer G. A. , Friedman H. , Reardon D. , Friedman A. , Bigner D. , Sampson J. H. 
Published: March 2020

2529 Background: Despite multimodality approaches, survival with GBM is dismal. Induction of immune responses to suppress the infiltrative, residual component with an easily manufactured and administered immunotherapy has been a theoretical ideal. The epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific cell surface protein expressed on approximately 40% of GBMs. Methods: Newly-diagnosed GBM patients with a gross-total resection, a KPS ≥70, and EGFRvIII+, after undergoing radiation with concurrent temozolomide without tumor progression, were eligible to receive EGFRvIII peptide vaccination i.d. with GM-CSF. Primary endpoint was safety. Results: Accrual began in 06/14/2004 and is now complete. 19 patients were enrolled. Median follow-up is 18 months. Toxicity was minimal and without evidence of autoimmunity. Humoral and cellular immune responses were generated. Median TTP from surgery in vaccine-treated patients is 12 months (n = 12), comparing favorably with a historical matched unvaccinated cohort (gross total resection without progression during radiation, KPS≥70, EGFRvIII+) that had a median TTP of 7.1 months (n = 39) (p = 0.0058). These results also compared favorably with those reported for concurrent temozolomide and radiation followed by adjuvant temozolomide, with a median TTP of 6.9 months. Median survival in this trial has exceeded 18 months which compares favorably to all published analyses accounting for all known prognostic indicators. Among recurrent tumors evaluated by immunohistochemistry, 100% no longer expressed the EGFRvIII, suggesting immunological activation that eliminated EGFRvIII-expressing cells, as well as one potential mechanism of treatment failure. Conclusions: EGFRvIII peptide vaccination warrants further investigation in a larger randomized clinical trial in patients with EGFRvIII-expressing tumors. No significant financial relationships to disclose.
Autologous tumor cell vaccination with PF-3512676 (CPG 7909) and GM-CSF followed by subcutaneous PF-3512676 and IFN-alpha for patients with metastatic renal cell carcinoma
Authors: Van Den Eertwegh A. J. , Lensen R. J. , Scheper R. J. , Giaccone G. , Meijer C. J. , Bontkes H. J. , Gruijl T. D. , Hooijberg E. 
Published: March 2020

2530 Background: Metastatic renal cell carcinoma (RCC) has a poor prognosis with a 5-year survival of less than 10%. Non-specific immunotherapy, e.g. IL-2 and IFN-α achieves responses in 10–30% of patients at the cost of relatively high toxicity. Tumor vaccination has shown great promise in a variety of cancers, particularly in the adjuvant setting. Studies in metastatic setting are suitable to identify the best adjuvants and to optimize vaccination schedules. CpGs are synthetic oligonucleotides that have been shown to have superior immunogenicity in animal and human vaccination studies (Hepatitis B). Besides its positive effect on the specific immune response, CpGs also stimulate the innate immune system. We initiated a phase I/II trial to investigate the potentials of CpG in the treatment of metastatic RCC. Methods: Nephrectomized patients with progressive metastatic RCC were vaccinated with autologous tumor cells (ATC) derived from the primary tumor or metastases. Vaccines consisted of irradiated ATC, PF-3512676 and GM-CSF. The first three induction vaccinations were given weekly followed by subcutaneous administration of IFN-α (6 MIU, TIW) and PF-3512676 (8 mg bi-weekly). After 3 months tumor evaluation was performed and in case of a remission or stable disease patients continued with 3 monthly vaccinations and PF-3512676 /IFN-α. Blood was collected for immunomonitoring and DTH responses against ATC were measured before and after vaccination. Results: Preparation of vaccines was successful in 88% of the patients. Twelve patients were included and treated according to the protocol. Treatment was well tolerated. At the vaccination site we observed erythema and induration. Some patients experienced flu-like symptoms, including fever and fatigue. In all patients we observed only after vaccination a DTH response (> 10 mm) against ATC, suggestive for a specific antitumor response. Three patients (25%) achieved a partial response (durations 6, 4+, 4+ months) and 2 patients (17%) stable disease (3 months). Conclusions: This new combined immunotherapy is tolerable, safe, and immunogenic. Moreover, it can produce tumor responses in advanced RCC. No significant financial relationships to disclose.
Impact of antibody Fc carbohydrate fucosylation on antibody-dependent cellular cytotoxicity by HuMax-EGFr —A fully human epidermal growth factor receptor antibody
Authors: Dechant M. , Lammerts van Bueren J. , Peipp M. , Bleeker W. , Beyer T. , Van Berkel P. , Parren P. W. , Van de Winkel J. G. , Valerius T. 
Published: March 2020

2543 Background: HuMax-EGFr - a human IgG1 EGF-R antibody in phase II clinical trials - was demonstrated to block EGF binding, to interfere with cellular signaling and to efficiently recruit effector cells for ADCC. Triggering of ADCC requires antibody binding to cellular Fc receptors which can be modulated by antibody glycosylation. Here, we investigated whether the glycosylation profile of HuMax-EGFr altered effector functions, and compared the contribution of mononuclear (MNC) and polymorphonuclear (PMN) cells for ADCC in human blood. Methods: Varying concentrations of two HuMax-EGFr batches, differing in the level of fucosylation of the complex-type oligosaccharides present in the Fc fragment, were compared in their capacity to kill EGF-R expressing tumor cells. Antibody binding and inhibition of proliferation was analyzed by flow cytometry and measuring vital cell mass, respectively. ADCC by isolated PMN, MNC and human whole blood was analyzed by chromium release assays. Results: Both batches of HuMax-EGFr bound similarly to target cells, and inhibited their proliferation to a similar extent. Low-fucosylated HuMax-EGFr demonstrated higher affinity for recombinant FcγRIIIa than fully fucosylated antibody. As expected, low-fucosylated antibody was significantly better in mediating ADCC by MNC (EC50 obtained at 10-fold lower antibody concentrations, p<0.05). In contrast, PMN-mediated ADCC was unaffected by the antibody fucosylation level. Importantly, whole blood ADCC assays also revealed little differences between both batches. ADCC was significantly enhanced by blood from G-CSF-primed patients, but again both high- and low-fucosylated antibody performed similarly. Conclusions: Both HuMax-EGFr batches were equally potent in inhibiting cell proliferation, and were highly active in triggering ADCC by MNC and PMN. Differences in fucosylation of Fc carbohydrate affected MNC-mediated lysis, whereas PMN- and whole blood-mediated cell killing were only minimally affected. Thus, fucosylation of antibody Fc may only prove critical for therapeutic antibodies which predominantly recruit NK cells for ADCC, while it may be less relevant for PMN-recruiting antibodies. [Table: see text]
Nonmyeloablative allogeneic blood stem cell transplantation (NMBSCT) ± donor lymphocyte infusion (DLI) for chemorefractory advanced nasopharyngeal carcinoma (NPC)
Authors: Toh H. C. , Tan E. H. , Thng C. H. , Yap S. P. , Lee K. M. , Phoon Y. P. , Chua S. , Soe Y. , Hee S. W. , Sun L. 
Published: March 2020

2549 Background: There is emerging clinical evidence for T cell immunotherapy in NPC. We report a NMBSCT regimen that induced mixed chimerism for DLI to potentially achieve a graft-versus-tumour effect (GVT) in NPC. Methods: 15 patients with advanced pretreated NPC were accrued to this prospective clinical trial. Our regimen comprised IV cyclophosphamide 50 mg/kg on day-5,-4,-3 and IV thymoglobuline on day-1, +1, +2 and +3 (n = 9) and then revised to IV thymoglobuline day-1, +1, +2, +3 (n = 6). Thymic radiation was given on day-1. G-CSF mobilized HLA-matched (n = 14) and 1-antigen-mismatched (n = 1) sibling PBSC were infused. DLI was initiated following taper of prophylactic cyclosporine (CyA). Results: Median age (n =15) was 49 yrs, median no. of metastatic organ sites was 3, mean no. of prior chemotherapy was 4 (range 2 - 8) and 14/15 had prior radiation. Median time to engraftment was 13 days and hospital stay was 17 days. Complete removal of prophylactic CyA was achieved for all patients at a median of day +28. DLI was given in 14 patients (median = 2, range 1 to 6). 4 patients were later re-started on GVHD immunosuppressive therapy. Two (13.3%) patients had acute GVHD (one Grade II and one fatal case who died on day+91). 3 patients developed chronic GVHD (20%), all controlled with short courses of standard immunosuppressive treatment. 11/15 died of PD, and 2/15 died from sepsis. 100-day TRM was 6.6%. 3/9 patients had donor graft loss. 6/6 patients on the revised protocol had stable and/or full donor chimerism. CMV antigenemia was 9/9 for the first regimen and 3/6 for the revised regimen (p = 0.044). Tumor response (RECIST) confirmed 40% PR, 33.3% SD and 40% PD, for a disease control rate of 73.3%. > day+100 anti-tumor response were observed in 5/15 patients (33.3%), implicating a GVT effect. 1 patient achieved a delayed PR on day+111 with stable donor chimerism of 19%. The longest surviving patient (36.5 mths) with multiple nodal, bone and lung metastasis achieved full donor chimerism and further tumor shrinkage at 8 mths post-NMBSCT. Conclusions: GVT is achievable with NMBSCT ± DLI in Stage IV NPC patients with a high disease control rate and acceptable GVHD. Early complete removal of prophylactic CyA was possible in all 15 patients. No significant financial relationships to disclose.
Metronomic cyclophosphamide regimen electively depletes CD4+ CD25+ regulatory T cells in patients with advanced solid tumors
Authors: Ghiringhelli Esq. F. , Ladoire S. , Zitvogel L. , Chauffert B. 
Published: March 2020

2561 Background: CD4+CD25+ regulatory T cells (Treg) are involved in the prevention of autoimmune diseases and in tumor-induced tolerance. We previously demonstrated in tumor-bearing rodents that low doses of cyclophosphamide (CPM) could significantly decrease both number and suppressive functions of Treg, facilitating vaccine-induced tumor rejection. Repeated low doses of cyclophosphamide, referred to as metronomic treatment aiming at reducing tumor angiogenesis, are used in patients with advanced, chemoresistant tumors. Methods: Six patients with metastatic solid tumors were treated after informed consent with metronomic CPM - 50 mg orally twice a day, with one week on, and one week off, until progression or limiting toxicity. Peripheral blood mononucleated cells (PBMC) were sampled before and after 4 weeks of treatment. Occurrence of blood leucocyte subpopulation and capacity of Treg to inhibit NK and T cells patients functions were measured. Results: metronomic CPM induced a profound and selective reduction of circulating Treg, both in percentage (7.7 ± 0.8 % before versus 3.3 ± 0.8 % after, p < 0.005) and absolute number (25.2 ± 7.5 cells/mm3 before versus 6.1 ± 3.3 cells/mm3 after, p < 0.0006). Inhibitory functions of Treg on conventional T cells and NK cells lead to a restoration of peripheral T cell proliferation - i.e. PBMC from cancer patients exhibited 17 ± 3% T cells proliferation capacity after CD3 CD28 stimulation versus 44 ± 5% (p < 0.01) 1 month after the beginning of the treatment, and versus 45 ± 6% in healthy volunteers- and innate NK killing activities - i.e. PBMC from cancer patients exhibited 9 ± 1% NK killing capacity against K562 target versus 26 ± 5% (p < 0.005) after 4 week treatment. Conclusions: Metronomic CPM has not only effect on tumor angiogenesis, but also strongly curtail immunosuppressive Treg, which could favor a better control of tumor progression. No significant financial relationships to disclose.
Adjuavnt tyrosinase peptide vaccination in patients with resected stage III/IV melanoma
Authors: Letsch A. , Scheibenbogen C. , Fluck M. , Asemissen A. , Nagorsen D. , Thiel E. , Keilholz U. 
Published: March 2020

2570 Background: This phase II study was undertaken to evaluate the immunogenicity and clinical efficacy of vaccination with tyrosinase peptides as adjuvant treatment in melanoma patients after multiple relapses. Methods: Stage III/IV melanoma patients after complete resection of relapses were vaccinated with tyrosinase peptides together with GM-CSF and KLH. Vaccination was given bi-weekly times 4, followed by monthly times 8 in absence of relevant disease progression. Tyrosinase-specific T cells were quantitated and characterized ex vivo by intracellular cytokine flow cytometry. Results: 44 patients were accrued with a median of 5.8 (range 2–25) previous relapses at cutaneous /s.c. only (n = 11), ln (n = 30) and/or visceral sites (n = 7). 11 patients received less than 6 vaccinations due to rapid progression. Of 32 patients evaluable for immunological response 16 (50%) displayed pre-vaccine spontaneous tyrosinase-specific T cells (median 0.32%). In 7 of the 15 patients without pre-existing T cells tyrosinase-specific T cells were induced after 6 vaccinations (median 0.08% CD3+CD8+ T cells) and in 10 of 12 patients after 12 vaccinations (median 0.36%, p = 0.03 compared to 6 vaccinations). Similarly, in 9 of the 16 patients with pre-existing T cell responses the frequency of tyrosinase-specific T cells did increase after 6 cycles (median 0.43 %), but in none of 10 patients with pre-existing T cells an increase was observed after 12 vaccinations (median 0.20%). Both, the spontaneous and the vaccine-induced T cells were predominantly of effector and effector-memory phenotype. With respect to clinical efficacy vaccination was terminated after 3 to 9 cycles due to disease progression in 19 of 44 patients. Of the remaining 25 patients receiving all 12 vaccinations 7 (16% of all 44 enrolled) had a favourable clinical course with cessation of recurrences. Conclusions: Taken together, this study shows high immunogenicity of the vaccine in patients with limited tumor burden associated with clinical efficacy. In case of a spontaneous pre-existing T cell response to the vaccine peptides, however, this could not, or only transiently be augmented. No significant financial relationships to disclose.