The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy

Zhang, Ying; Sultonova, Rukhsora D.; You, Sung-Hwan; Choi, Yoonjoo; Kim, Soyoung; Lee, Wan‐Sik; Seong, Jihyoun; Min, Jung‐Joon; Hong, Yeongjin

doi:10.1016/j.bcp.2023.115473

Cited by 10 publications

(10 citation statements)

References 73 publications

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“…E. coli BL21(DE3) strains transformed with expression plasmids (pETh-CRT3-LASP-PAS200, pETh-CRT4-LASP-PAS200, or pETh-Fn3(DGR)-LASP-PAS200) were used to purify PASylated CRT-targeting L-ASNases (CRT3LP, CRT4LP, or #DGRLP) as described 49 . E. coli BL21(DE3) strains transformed with expression plasmids pETh-CRT3-Rluc8, pETh-CRT4-Rluc8, or pETh-Fn3(DGR)-Rluc8 were used to purify Renilla luciferase variant 8 (Rluc8)-conjugated monobodies (CRT3-Rluc8, CRT4-Rluc8, or #DGR-Rluc8) as described 48 .…”

Section: Methodsmentioning

confidence: 99%

“…E. coli BL21(DE3) strains transformed with expression plasmids pETh-CRT3-Rluc8, pETh-CRT4-Rluc8, or pETh-Fn3(DGR)-Rluc8 were used to purify Renilla luciferase variant 8 (Rluc8)-conjugated monobodies (CRT3-Rluc8, CRT4-Rluc8, or #DGR-Rluc8) as described 48 . The conjugated proteins were expressed in the above bacterial strains and purified using His GraviTrap columns (GE Healthcare, USA) and PD-10 desalting column (GE Healthcare, USA), as previously described 49 . Endotoxin contamination was removed from the purified proteins using high-capacity endotoxin removal spin columns (Thermo Fisher Scientific, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Endotoxin contamination was removed from the purified proteins using high-capacity endotoxin removal spin columns (Thermo Fisher Scientific, USA). Finally, protein concentration and ASNase activity were measured with an asparaginase activity assay kit and an OxiRed probe (Abcam, USA) 49 . The specific activity of CRT-targeting L-ASNases was similar to that published previously (i.e., ∼6.5 IU/nmol of each monobody) 49 .…”

Section: Methodsmentioning

confidence: 99%

“…Finally, protein concentration and ASNase activity were measured with an asparaginase activity assay kit and an OxiRed probe (Abcam, USA) 49 . The specific activity of CRT-targeting L-ASNases was similar to that published previously (i.e., ∼6.5 IU/nmol of each monobody) 49 .…”

Section: Methodsmentioning

confidence: 99%

“…These monobodies specifically target ecto-CRT in solid tumors of mice treated with ICD-inducing chemotherapy. Additionally, we generated PASylated L-ASNases conjugated with CRT-specific monobodies (CRT3LP and CRT4LP), which increased the therapeutic efficacy of chemotherapy 49 . Here, we investigated whether CRT-targeting L-ASNases can increase the therapeutic efficacy of RT by activating immune responses.…”

Section: Introductionmentioning

confidence: 99%

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The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy

Zhang,

Akhil,

Seo

et al. 2024

Theranostics

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Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4 + T, and CD8 + T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy

Zhang,

Akhil,

Seo

et al. 2024

Theranostics

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l‐Asparaginase Bio‐Betters: Insight Into Current Formulations, Optimization Strategies and Future Bioengineering Frontiers in Anti‐Cancer Drug Development

Sonowal,

Pathak,

Das

et al. 2024

Advanced Therapeutics

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Cancer remains a persistent global health concern, representing a significant challenge in medical science and patient care. In this context, l‐asparaginase has emerged as a promising therapeutic agent due to its unique ability to deplete circulating asparagine, thereby selectively targeting cancer cells. However, despite its potential, current formulations of l‐asparaginase are not without limitations. Issues such as immunogenicity, short half‐life, and variable efficacy present hurdles in its widespread clinical application. To overcome these hurdles, researchers are focusing on developing bio‐better versions of l‐asparaginase. These bio‐betters aim to enhance stability, reduce immunogenicity, and optimize enzyme kinetics, thus improving treatment outcomes. This review critically assesses the current landscape of l‐asparaginase bio‐betters, offering insights into ongoing formulations and advancements, optimization strategies, and future bio‐engineering frontiers. It discusses modifications to enhance therapeutic properties and explores innovative approaches like in‐silico enzyme engineering and artificial intelligence, highlighting their potential to improve the therapeutic profile of l‐asparaginase. Challenges and debates surrounding the l‐asparaginase mechanism are also addressed. By addressing current challenges and outlining future directions, this review aims to contribute to the advancement of anti‐cancer therapeutics, particularly in the context of l‐asparaginase bio‐better research.

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NIR Enhanced pH‐Responsive Microneedles for Synergetic Therapy of Melanoma

Han,

Yu,

Liu

et al. 2024

ChemMedChem

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Melanoma has emerged as a significant threat to human life and health. Microneedle (MN)‐mediated transdermal drug delivery (TDD) has garnered attention in melanoma treatment for bypassing the first‐pass effect. However, the propensity of melanoma to metastasize presents substantial challenges for MN mediated local treatment. Developing systemic therapies, such as immunotherapy in combination with TDD, is crucial for achieving effective melanoma treatment. Herein, a polyvinyl alcohol (PVA) MN‐mediated multifunctional TDD system, designated MN@PDA@1‐MT/CUR/DOX@HA (MN@PMCDH), was developed for synergetic chemotherapy/photothermal/immunotherapy of melanoma. PMCDH nanomedicines penetrate deep skin layers through MNs, accumulate at tumor sites guided by hyaluronic acid (HA), and selectively release drugs in response to the acidic tumor microenvironment and near‐infrared (NIR) stimulation. Released curcumin (CUR) significantly enhances the efficacy of photothermal therapy (PTT) and chemotherapy, as well as improves the induction of immunogenic cell death (ICD) by increasing melanoma sensitivity to polydopamine (PDA)‐mediated photothermal effects and doxorubicin (DOX). Moreover, the incorporation of 1‐methyltryptophan (1‐MT) to reverse the tumor immunosuppressive microenvironment can further enhance the effects of immunotherapy. In vitro studies revealed that the MN@PMCDH system can effectively induce ICD and inhibit tumor cell growth. Additionally, remarkable deep tumor cell inhibition effects are also achieved in 3D tumor models.

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The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy

Cited by 10 publications

References 73 publications

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy

l‐Asparaginase Bio‐Betters: Insight Into Current Formulations, Optimization Strategies and Future Bioengineering Frontiers in Anti‐Cancer Drug Development

NIR Enhanced pH‐Responsive Microneedles for Synergetic Therapy of Melanoma

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