Acquired resistance to sunitinib is a challenge in the treatment of renal cell carcinoma (RCC). The dysregulation of cellular metabolism is prevalent during resistance acquisition. It is known that in sunitinib-resistant RCC 786-O (786-O Res) cells sunitinib is mainly sequestered in the intracellular lysosomes. However, the relevance between sunitinib resistance and cellular metabolism has not been examined. In this study, we examined the metabolic changes in 786-O Res by using capillary electrophoresis-time of flight mass spectrometry. The cell line 786-O Res was established via persistent treatment with sunitinib, where increase in intracellular sunitinib, and sizes of lysosomes and nuclei were enhanced as compared with those in the parental 786-O (786-O Par) cells. Metabolic analyses revealed that out of the 110 metabolites examined, 13 were up-regulated and 4 were down-regulated in the 786-O Res cells. The glycolysis, tricarboxylic acid cycle and pentose phosphate pathway (PPP) were identified as being altered in the sunitinib-resistant cells, which resulted in the enhanced metabolisms of energy, nucleic acids, and glutathione redox cycle. As sunitinib was sequestered in the enlarged lysosomes in 786-O Res, the enriched energy metabolism might contribute to the maintenance of luminal pH in lysosomes via the H+ ATPase. The changes in the PPP could contribute to nuclei enlargement through up-regulation of nucleic acid biosynthesis and protect 786-O Res from cytotoxicity induced by sunitinib through up-regulation of reduced glutathione. Though the direct link between sunitinib resistance and metabolic alternation remains to be elucidated, this metabolomics study provides fundamental insights into acquisition of sunitinib resistance.
BackgroundRecently, antiprogrammed cell death protein 1 (aPD-1) and antiprogrammed death-ligand 1 (aPD-L1) monoclonal antibodies (mAbs) have been approved. Even though aPD-1 and aPD-L1 mAbs target the same PD-1/PD-L1 axis, it is still unclear whether both mAbs exert equivalent pharmacological activity in patients who are sensitive to PD-1/PD-L1 blockade therapy, as there is no direct comparison of their pharmacokinetics (PK) and antitumor effects. Therefore, we evaluated the differences between both mAbs in PK and therapeutic effects in PD-1/PD-L1 blockade-sensitive mouse models.MethodsHerein, murine breast MM48 and colon MC38 xenografts were used to analyze the pharmacological activity of aPD-1 and aPD-L1 mAbs. The PK of the mAbs in the tumor-bearing mice was investigated at low and high doses using two radioisotopes (Indium-111 and Iodine-125) to evaluate the accumulation and degradation of the mAbs.ResultsaPD-1 mAb showed antitumor effect in a dose-dependent manner, indicating that the tumor model was sensitive to PD-1/PD-L1 blockade therapy, whereas aPD-L1 mAb failed to suppress tumor growth. The PK study showed that aPD-L1 mAb was accumulated largely in normal organs such as the spleen, liver, and kidney, resulting in low blood concentration and low distributions to tumors at a low dose, even though the tumors expressed PD-L1. Sufficient accumulation of aPD-L1 mAb in tumors was achieved by administration at a high dose owing to the saturation of target-mediated binding in healthy organs. However, degradation of aPD-L1 mAb in tumors was greater than that of aPD-1 mAb, which resulted in poor outcome presumably due to less inhibition of PD-L1 by aPD-L1 mAb than that of PD-1 by aPD-1 mAb.ConclusionAccording to the PK studies, aPD-1 mAb showed linear PK, whereas aPD-L1 mAb showed non-linear PK between low and high doses. Collectively, the poor PK characteristics of aPD-L1 mAb caused lower antitumor activity than of aPD-1 mAb. These results clearly indicated that aPD-L1 mAb required higher doses than aPD-1 mAb in clinical setting. Thus, targeting of PD-1 would be more advantageous than PD-L1 in terms of PK.
BackgroundWith the increased use of immune checkpoint inhibitors (ICIs), side effects and toxicity are a great concern. Anaphylaxis has been identified as a potential adverse event induced by ICIs. Anaphylaxis is a life-threatening medical emergency. However, the mechanisms and factors that can potentially influence the incidence and severity of anaphylaxis in patients with cancer remain unclear.MethodsHealthy, murine colon 26, CT26, breast 4T1, EMT6, and renal RENCA tumor-bearing mice were treated with an anti-PD-L1 antibody (clone 10F.9G2). Symptoms of anaphylaxis were evaluated along with body temperature and mortality. The amounts of antidrug antibody and platelet-activating factor (PAF) in the blood were quantified via ELISA and liquid chromatography-mass spectrometry (LC-MS/MS). Immune cells were analyzed and isolated using a flow cytometer and magnetic-activated cell sorting, respectively.ResultsRepeated administration of the anti-PD-L1 antibody 10F.9G2 to tumor-bearing mice caused fatal anaphylaxis, depending on the type of tumor model. After administration, antidrug immunoglobulin G (IgG), but not IgE antibodies, were produced, and PAF was released as a chemical mediator during anaphylaxis, indicating that anaphylaxis was caused by an IgG-dependent pathway. Anaphylaxis induced by 10F.9G2 was treated with a PAF receptor antagonist. We identified that neutrophils and macrophages were PAF-producing effector cells during anaphylaxis, and the tumor-bearing models with increased numbers of neutrophils and macrophages showed lethal anaphylaxis after treatment with 10F.9G2. Depletion of both neutrophils and macrophages using clodronate liposomes prevented anaphylaxis in tumor-bearing mice.ConclusionsThus, increased numbers of neutrophils and macrophages associated with cancer progression may be risk factors for anaphylaxis. These findings may provide useful insights into the mechanism of anaphylaxis following the administration of immune checkpoint inhibitors in human subjects.
Objective: Recently anti-PD-1 antibodies (aPD-1 Abs), anti-PD-L1 (aPD-1) Abs have been approved. However, the difference between both Abs in pharmacokinetics and anti-tumor effects have not been fully understood. In this study, we analyzed the difference between both Abs in blood concentration, biodistribution and degradation in tumor-bearing mice by using aPD-1/PD-L1 Abs labeled with radioisotopes (In-111/I-125) and evaluated the relationship between PK and therapeutic effects. Method: Abs were labeled with In-111 via chelate agents, and labeled with I-125 through covalent bond. Tumor bearing mice were prepared by s.c. inoculation with mouse colon cancer MC38 cells or mouse breast cancer MM48 cells. The labeled Abs were intraperitoneally injected into tumor-bearing mice. Tumors and organs were harvested at several time points after the injection, and radio activities in organs were measured by a gamma counter. The accumulation of Abs were expressed as % of injected dose/g organs. Because In-111 tends to be accumulated in organs due to poor permeability and I-125 was eliminated from organs rapidly due to high permeability, the ratio of I-125 and In-111 could reflect the degradation of Abs after cellular uptake. In pharmacological studies, Abs were intraperitoneally injected into tumor-bearing mice at doses of 50 to 200 μg (2.5 to 10 mg/kg) at day 5, 8, and 12 after tumor-inoculation. Tumor volume was evaluated to evaluate tumor progression. Result and Discussion: aPD-1 Ab showed anti-tumor effect in both MC38 and MM48 tumor models in a dose-dependent manner. On the other hand, aPD-L1 Abs showed lower anti-tumor effect in MC38 models, and negligible effect in MM48 tumor bearing mice at tested doses. According to PK studies, it was observed that aPD-L1 Abs were largely accumulated in normal tissues, especially in the spleen, liver, and kidney, and degraded rapidly compared with aPD-1 Abs, resulting that the blood concentration and distribution in tumors of aPD-L1 Abs tended to be low. Moreover, aPD-L1 Abs showed more rapid degradation in both tumors than aPD-1 Ab. Conclusion: aPD-L1 Ab showed less anti-tumor effect in tested tumor models due to less distribution and faster degradation in tumors than aPD-1 Ab. Collectively, the PK of aPD-1/PD-L1 Abs which target the same axis were not equivalent and the selectivity of expression of target molecules in both normal tissues and tumors should be considered to optimize their therapeutic efficacy. Citation Format: Hiroto Hatakeyama, Taiki Kurino, Reiko Matsuda, Hiroyuki Suzuki, Ayu Terui, Tomoya Uehara, Yasushi Arano, Akihiro Hisaka. Pharmacokinetic analysis of anti-PD-1 and PD-L1 antibodies and evaluation of their anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3232.
Introduction: Anti-PD-1 antibodies (αPD-1 Abs) are currently used in cancer immunotherapy that block the interaction between PD-1 and PD-L1. In addition to anti-PD-1 Abs, anti-PD-L1 (αPD-L1) Abs have also been approved to inhibit the PD-1/PD-L1 axis. However, the difference between both Abs in pharmacokinetics and anti-tumor effects have not been fully understood. In this study, we analyzed the difference between both Abs in blood concentration, biodistribution and degradation in tumor-bearing mice by using αPD-1/PD-L1 Abs labeled with radioisotopes (111In/125I) and evaluated the relationship between PK and therapeutic effects. Methods: Abs were labeled with 111In via chelate agents, and labeled with 125I through covalent bond. Tumor bearing mice were prepared by s.c. inoculation with mouse colon cancer MC38 cells or mouse breast cancer MM48 cells. The labeled Abs were intraperitoneally injected into tumor-bearing mice. Tumors and organs were harvested at several time points after the injection, and radioactivities in organs were measured by a gamma counter. The accumulation of Abs was expressed as % of injected dose/g organs. Because 111In tends to be accumulated in organs due to poor permeability and 125I was eliminated from organs rapidly due to high permeability, the ratio of 125I and 111In could reflect the degradation of Abs after cellular uptake. In pharmacologic studies, Abs were intraperitoneally injected into tumor-bearing mice at day 5, 8, and 12 after tumor-inoculation. Tumor volume was evaluated to evaluate tumor progression. Results and Discussion: It was observed that αPD-L1 Ab was largely accumulated in normal tissues, especially in the spleen and liver and degraded rapidly compared with αPD-1 Ab, resulting that the blood concentration and distribution in tumors of αPD-L1 Ab tended to be low in both tumor-bearing mice models. Moreover, αPD-L1 Ab showed lower antitumor effect due to less delivered aPD-L1 Ab to tumors than aPD-1 Ab. Collectively, the PK of αPD-1/PD-L1 Abs that target the same axis were not equivalent and the selectivity of expression of target molecules in both normal tissues and tumors should be considered to optimize their therapeutic efficacy. Citation Format: Hiroto Hatakeyama, Taiki Kurino, Hiroyuki Suzuki, Ayu Terui, Tomoya Uehara, Yasushi Arano, Akihiro Hisaka. Comparative analysis of pharmacokinetics and antitumor effect between anti-PD-1 and anti-PD-L1 in mice models [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B160.
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