Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody

XU Shuyi; LI Yaxian; HU Hai; ZHANG Li; BIAN Yanlin; ZHU Jianwei; WU Mingyuan

doi:10.11665/j.issn.1000-5048.20210516

Journal of China Pharmaceutical University > 2021 > 52(5): 622-629. > DOI: 10.11665/j.issn.1000-5048.20210516

XU Shuyi, LI Yaxian, HU Hai, ZHANG Li, BIAN Yanlin, ZHU Jianwei, WU Mingyuan. Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody[J]. Journal of China Pharmaceutical University, 2021, 52(5): 622-629. DOI: 10.11665/j.issn.1000-5048.20210516

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Purification and activity of anti-PD-L1&CXCR4 bispecific nanobody

1.
Engineering Research Center of Cell and Therapeutic Antibody (MOE),School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240
2.
Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China

Funds: This study was supported by the National Nature Science Foundation of China (No.81773621) and the National Facility for Translational Medicine (Shanghai) Open Research Program (No.TMSK-2020-131)

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Received Date: March 01, 2021
Revised Date: September 25, 2021

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Abstract

Abstract

Targeted programmed death-ligand 1 (PD-L1) and CXC chemokine receptor type 4 (CXCR4), gene sequences encoding anti-PD-L1 nanobody and anti-CXCR4 nanobody were cloned into the pET-22b (+) vector to construct recombinant expression plasmid of anti-PD-L1&CXCR4 bispecific nanobody, which was connected with 6 × His tag and transformed into E.coli BL21 (DE3). The expressed proteins were then found to exist as a soluble form in the supernatant of bacterial lysate after induction of IPTG.Three purification methods were used to obtain the target protein in order to improve the yield and purity of the bispecific nanobody.The bacterial supernatant was separated and purified by His Trap FF affinity chromatographic column.The target protein output could exceed 1 mg/L, and the product purity could reach up to 97%.Besides, the anti-PD-L1&CXCR4 bispecific nanobody shows a specific binding ability to two antigens on the cell surface, enhancing the cytotoxicity of IL-2 activated human peripheral blood mononuclear cells (PBMC) to tumor cell line AsPC-1, which lays the foundation for further evaluation of its drug efficacy in vivo.
- bispecific,
- nanobody,
- affinity chromatography,
- protein purification,
- biological activity

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[1]	. Nanomedicine （Lond），2015，10（1）：161-174.
[2]	Revets H，De Baetselier P，Muyldermans S. Nanobodies as novel agents for cancer therapy［J］. Expert Opin Biol Ther，2005，5（1）：111-124.
[3]	Fernandes JC. Therapeutic application of antibody fragments in autoimmune diseases：current state and prospects［J］. Drug Discov Today，2018，23（12）：1996-2002.
[4]	Roovers RC，Vosjan MJ，Laeremans T，et al. A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth［J］. Int J Cancer，2011，129（8）：2013-2024.
[5]	Peyvandi F，Scully M，Kremer Hovinga JA，et al. Caplacizumab for acquired thrombotic thrombocytopenic Purpura［J］. N Engl J Med，2016，374（6）：511-522.
[6]	Xing XY，Wang XC，He W. Advances in research on tumor immunotherapy and its drug development［J］. J China Pharm Univ（中国药科大学学报），2021，52（1）：10-19.
[7]	Yang J，Hu LQ. Immunomodulators targeting the PD-1/PD-L1 protein-protein interaction：from antibodies to small molecules［J］. Med Res Rev，2019，39（1）：265-301.
[8]	Asano T，Meguri Y，Yoshioka T，et al. PD-1 modulates regulatory T-cell homeostasis during low-dose interleukin-2 therapy［J］. Blood，2017，129（15）：2186-2197.
[9]	Zou W，Wolchok JD，Chen L. PD-L1 （B7-H1） and PD-1 pathway blockade for cancer therapy：mechanisms，response biomarkers，and combinations［J］. Sci Transl Med，2016，8（328）：328rv4.
[10]	Nowicki TS，Hu-Lieskovan S，Ribas A. Mechanisms of resistance to PD-1 and PD-L1 blockade［J］. Cancer J，2018，24（1）：47-53.
[11]	Anderson KG，Stromnes IM，Greenberg PD. Obstacles posed by the tumor microenvironment to T cell activity：a case for synergistic therapies［J］. Cancer Cell，2017，31（3）：311-325.
[12]	Domanska UM，Kruizinga RC，Nagengast WB，et al. A review on CXCR4/CXCL12 axis in oncology：no place to hide［J］. Eur J Cancer，2013，49（1）：219-230.
[13]	Scala S. Molecular pathways：targeting the CXCR4-CXCL12 axis：untapped potential in the tumor microenvironment［J］. Clin Cancer Res，2015，21（19）：4278-4285.
[14]	Bockorny B，Semenisty V，Macarulla T，et al. BL-8040，a CXCR4 antagonist，in combination with pembrolizumab and chemotherapy for pancreatic cancer：the COMBAT trial［J］. Nat Med，2020，26（6）：878-885.
[15]	Abraham M，Mishalian I，Harel Y，et al. Effect of BL-8040，high-affinity CXCR4 antagonist，on T-cell infiltration，tumor growth，and synergy with immunomodulatory agents［J］. J Clin Oncol，2017，35（15_suppl）：e14544.
[16]	Li ZT，Wang YX，Shen YX，et al. Targeting pulmonary tumor microenvironment with CXCR4-inhibiting nanocomplex to enhance anti-PD-L1 immunotherapy［J］. Sci Adv，2020，6（20）：eaaz9240.
[17]	Zhang F，Wei HD，Wang XX，et al. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade［J］. Cell Discov，2017，3：17004.
[18]	J?hnichen S，Blanchetot C，Maussang D，et al. CXCR4 nanobodies （VHH-based single variable domains） potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells［J］. PNAS，2010，107（47）：20565-20570.
[19]	Yang WH，Chen PW，Li HC，et al. PD-L1：PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro［J］. Invest Ophthalmol Vis Sci，2008，49（6）：2518-2525.
[20]	Suurs FV，Lub-de Hooge MN，de Vries EGE，et al. A review of bispecific antibodies and antibody constructs in oncology and clinical challenges［J］. Pharmacol Ther，2019，201：103-119.
[21]	Qin WT，Hu LP，Zhang XL，et al. The diverse function of PD-1/PD-L pathway beyond cancer［J］. Front Immunol，2019，10：2298.
[22]	Seo YD，Jiang XY，Sullivan KM，et al. Mobilization of CD8⁺ T cells via CXCR4 blockade facilitates PD-1 checkpoint therapy in human pancreatic cancer［J］. Clin Cancer Res，2019，25（13）：3934-3945.
[23]	Decrop W，Swart R，Gendeh G. Development of an automated method for monoclonal antibodies purification and analysis［J］. J Biomol Tech，2010，21（3 suppl）：S34-S35.

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