Therapeutic anti-NLGP monoclonal antibody for carcinoembryonic antigen expressing tumors is nontoxic to Swiss and BALB/c mice.

A murine monoclonal antibody (mAb), 1C8 was developed against a novel glycoprotein NLGP and its unique property to recognize carcinoembryonic antigen (CEA) was reported. Utilizing this CEA recognizing property, 1C8 is successful to restrict the growth of CEA(+) murine and human cancers both in vitro and in vivo. Here, we have thoroughly evaluated the toxicity profile of this mAb 1C8 on different physiological systems of both tumor-free and tumor-bearing Swiss and BALB/c mice. Effective concentration (25 µg/mice) of 1C8 caused no behavioral changes in animals and no death was recorded. Moreover, little increase in the body and organ weights in all mice groups was noted. MAb 1C8 showed no adverse effect on the hematological system, but little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine assessment confirmed no abnormalities in both hepatic and renal functions. Number of T cells, B cells, NK cells, macrophages and dendritic cells was upregulated in vivo by mAb treatment with significant downregulation of regulatory T cells. During this treatment serum levels of type 1 cytokines were upregulated over type 2 cytokines. This mAb 1C8 also did not induce any significant increase in antibody titer following treatment. Accumulated evidences from Swiss and BALB/c mice strongly suggest that this mAb 1C8 is completely safe, thus, can be recommended for further clinical trial for the therapy of CEA(+) tumors.

Novel swine model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available. STUDY DESIGN AND METHODS: To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 µg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2 /FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted. RESULTS: Lung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p < 0.05). Bilateral infiltration was shown in chest X-ray imaging. Lung injury was confirmed by histopathologic analysis. CONCLUSION: Lung injury in pigs was successfully induced by anti-SLA MoAb. Priming with LPS is a prerequisite for inducing lung injury and the amount of the antibody is a critical condition.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

[Chemotherapy-free treatment of chronic lymphocytic leukemia?]. / Chemotherapie-freie Behandlung der chronischen lymphatischen Leukämie?

Treatment of CLL patients with conventional cytotoxic agents is often combined with significant toxicity that prevents broad application especially in elderly patients. In addition, relapse frequently occurs after application of conventional chemotherapy in CLL. Recently several new chemo-free treatment options have been introduced within clinical trials. Among them are monoclonal antibodies, most of them targeting the CD20 molecule: besides the licensed drugs rituximab and ofatumumab obinutuzumab, although in combination with chemotherapy, has recently shown high clinical efficacy in front-line treatment of elderly patients with CLL. Lenalidomide as monotherapy has demonstrated clinical efficacy in patients with relapsed disease and first data within clinical trials have been generated in the front-line setting. A promising class of novel agents has been designed to block aberrant signaling from the B-cell receptor. Ibrutinib acts by inhibiting the Bruton's tyrosine kinase (BTK) while idelalisib represents a first-in-class specific inhibitor of the phosphoinositol-3 kinase (PI3K) delta isoform. Another class of drugs with potential impact for chemo-free treatment strategies in CLL are the BH3-mimetic inhibitors of the Bcl-2 family of pro-survival proteins. Other interesting candidate drugs that are currently explored for CLL patients include small modular immunopharmaceutical (SMIP) proteins (e. g. TRU-016), CDK inhibitors (e. g. dinaciclib), HDAC inhibitors and others. Given all these novel agents and targets, chemo-free or at least chemo-reduced concepts may become reality in the near future for our patients suffering from CLL.

Retinal toxicity of intravitreal rituximab in albino rabbits.

PURPOSE: To evaluate retinal toxicity of intravitreal rituximab. METHODS: Twelve albino rabbits were included in the study. 1 mg/0.1 mL of rituximab was injected to the right (experimental) eye of each rabbit, and 0.1 mL of saline was injected into the left (control) eye. Electroretinogram was recorded before injection and 3 hours, 4 days, 1 week, 2 weeks and 4 weeks after injection (12 rabbits), and visual evoked potential was recorded before injection and 4 weeks after injection (10 rabbits). Histology and glial fibrillary acidic protein immunocytochemistry (12 rabbits) were performed at 4 weeks after injection. Clinical examination was conducted at all time points in all rabbits. RESULTS: The average dark-adapted electroretinogram b-wave Vmax ratios, and the average light-adapted b-wave amplitude ratios were approximately 1, and the average log σ difference was around zero throughout the follow-up period. The average visual evoked potential amplitude ratio and the average visual evoked potential implicit time difference were approximately 1 and 0, respectively. No histologic damage was seen, but glial fibrillary acidic protein was mildly expressed in 6 of 12 experimental eyes. Results of clinical examination were normal in all the eyes. CONCLUSION: Intravitreal injection of 1 mg rituximab does not cause functional or histologic signs of retinal toxicity in albino rabbits. Mild glial fibrillary acidic protein expression in Müller cells probably indicates a mild degree of retinal stress.

Long-term stability of diluted solutions of the monoclonal antibody rituximab.

As it is an important challenge for pharmacists to access the stability of monoclonal antibodies because of the widespread of centralized preparation units, we conducted a study to evaluate the physicochemical and biological stability of diluted rituximab at 1mg/mL over six months at 4 °C. We also conducted the study at 40 °C to demonstrate that all methods employed were stability indicating. Various protein characterization methods were used to determine changes in physicochemical properties of rituximab, including size-exclusion chromatography, dynamic light scattering, turbidimetry, cation-exchange chromatography, second-derivative ultraviolet and infrared spectroscopy, and peptide mapping. Cell culture was used to assess biological stability. We demonstrated that diluted rituximab stored at 4 °C in polyolefine bags remained stable for at least six months. No physical or chemical instability was observed, and the biological activity was fully maintained. Size exclusion chromatography did not show polymerization or fragmentation. No difference was noticed in the hydrodynamic diameter of RTX. No additional peak or decrease in the areas under curve was found by cation exchange chromatography. The thermal aggregation curves and their derived thermodynamic parameters were unchanged. The primary, secondary and tertiary structures of the protein were not modified. Finally, the direct cytotoxic effect of rituximab was fully maintained.

Preclinical evaluation of 227Th-labeled and 177Lu-labeled trastuzumab in mice with HER-2-positive ovarian cancer xenografts.

OBJECTIVE: The aim of the present study was to compare the biodistribution, normal tissue toxicity, and therapeutic effect of two low-dose rate radioimmunoconjugates (RICs) in mice with HER2-expressing ovarian cancer xenografts: the α-particle-emitting (227)Th-trastuzumab and the ß-particle-emitting (177)Lu-trastuzumab. MATERIALS AND METHODS: Trastuzumab (Herceptin), conjugated to DOTA and radiolabeled with (227)Th or (177)Lu, was injected intravenously into mice bearing SKOV-3 xenografts. The biodistribution was determined at different time points after injection. The organs were collected and measured for radioactivity content using a gamma spectrometer. Inhibition of tumor growth was measured after a single injection of (227)Th-trastuzumab, (227)Th-rituximab, (177)Lu-trastuzumab, trastuzumab alone, and NaCl. The toxicity of (227)Th-trastuzumab and (177)Lu-trastuzumab was evaluated by measurement of body weight, determination of blood cell counts, analysis of clinical chemistry parameters, and histological examination of tissue specimens. RESULTS: The absorbed radiation dose to the tumor was 4 Gy after administration of 400 kBq/kg (227)Th-trastuzumab and 72 MBq/kg (177)Lu-trastuzumab. A significantly better antitumor effect of (227)Th-trastuzumab (8 and 30 days' growth delay for 400 and 600 kBq/kg, respectively) was observed as compared with untreated control, trastuzumab alone, 600 kBq/kg (227)Th-rituximab (nonspecific targeting), and 72 MBq/kg (177)Lu-trastuzumab. Mean survival of mice after treatment with (227)Th-trastuzumab (107 ± 9 and 129 ± 12 days for 400 and 600 kBq/kg (227)Th-trastuzumab, respectively) was significantly improved compared with control (88 ± 11 days) and other RICs (85 ± 8 and 66 ± 6 days for 72 MBq/kg (177)Lu-trastuzumab and 600 kBq/kg (227)Th-rituximab, respectively) (P<0.05, Kaplan-Meier). Treatment-related toxicity was not observed in any group except for a transient decrease in white blood cells between 3 and 9 weeks after treatment with 400 and 600 kBq/kg (227)Th-trastuzumab. CONCLUSION: The α-particle-emitting RIC (227)Th-trastuzumab effectively delayed tumor growth and prolonged survival of mice compared with ß-emitting (177)Lu-trastuzumab administered at the same absorbed radiation dose to tumor. This new therapeutic approach warrants further studies aiming at clinical testing in patients with micrometastatic ovarian cancer.

Dose-adjusted EPOCH-rituximab combined with fludarabine provides an effective bridge to reduced-intensity allogeneic hematopoietic stem-cell transplantation in patients with lymphoid malignancies.

PURPOSE: There is currently no standard chemotherapy regimen for patients with lymphoid malignancies being considered for reduced-intensity conditioning allogeneic hematopoietic stem-cell transplantation (RIC-alloHSCT). The ideal regimen would provide disease control and result in lymphocyte depletion to facilitate engraftment. To this end, we developed a novel regimen by adding fludarabine to dose-adjusted continuous-infusion etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin plus with or without rituximab (DA-EPOCH-F/R). PATIENTS AND METHODS: One hundred forty-seven patients with lymphoid malignancy (median age, 50 years) who had heavily pretreated (median prior regimens, three) and chemo-refractory (47%) disease were treated with DA-EPOCH-F/R before RIC-alloHSCT. Patients received one to three consecutive cycles until achieving lymphocyte depletion (CD4(+) count < 200/µL) or progressive disease. RESULTS: Overall response rate was 41%; 39% of patients had stable disease. Toxicity included grade 4 neutropenia in 65% and thrombocytopenia in 25% of patients. DA-EPOCH-F/R resulted in lymphocyte depletion (P < .001), which was inversely associated with serum interleukin (IL) 7 and IL-15 levels. Of 147 patients, 143 patients proceeded to RIC-alloHSCT. Patients with lower CD3(+) (P < .001), CD4(+) (P < .001), and CD8(+) (P < .001) T-cell counts after DA-EPOCH-F/R were more likely to achieve full donor lymphoid chimerism by day +14 after transplant. Relative to nonresponders to DA-EPOCH-F/R, patients with complete and partial response had increased event-free survival (77.4 v 4.8 months; P < .001) and overall survival (98.5 v 16.2 months; P < .001). CONCLUSION: DA-EPOCH-F/R safely provides tumor cytoreduction and lymphocyte depletion, thereby offering a bridge to RIC-alloHSCT in patients with aggressive lymphoid malignancies.

Developmental immunotoxicology assessment of rituximab in cynomolgus monkeys.

Rituximab is a chimeric murine/human-engineered immunoglobulin (Ig) G1 anti-CD20 monoclonal antibody, selectively depleting CD20-expressing cells in peripheral blood and lymphoid tissues. As part of the rituximab registration-enabling program for rheumatoid arthritis, cynomolgus monkey embryo-fetal development and pre- and postnatal developmental toxicity studies were performed. In both studies, female cynomolgus monkeys were administered rituximab iv at doses of 0/0, 15/20, 37.5/50, and 75/100 mg/kg (loading dose/study dose) from gestation day (GD) 20 to 50 for the embryo-fetal development study and GD 20 to postpartum (pp) day 28 for the pre- and postnatal study. In the embryo-fetal development study, although maternal dosing ended during the first trimester at GD 50, placental transfer of rituximab to fetuses was demonstrated at GD 100. Consequently, fetuses demonstrated B-cell depletion in lymphoid tissues at GD 100. Repletion of B cells was demonstrated in infants in a follow-up pre- and postnatal study following fetal and neonatal exposure. In the pre- and postnatal study, despite B-cell depletion, there was no significant functional consequence on the infant's ability to mount T-cell-dependent antibody responses following vaccination or antigenic challenge. Overall, rituximab was well tolerated at maximum feasible doses up to 100 mg/kg in pregnant cynomolgus monkeys and their infants after exposure from the period of organogenesis throughout pregnancy, parturition, and postnatal development. Importantly, the preclinical data have been concordant with the clinical data in children for cases where rituximab was administered during pregnancy.