Expression of extracellular fragment of murine PD-L1 and production of antibodies to PD-L1

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Abstract

A number of molecules expressed on mammalian cells are involved in the formation of autotolerance. These primarily include CTLA-4/B7 and PD1-PD-L1 signaling pathways. Blockers of these signaling pathways, called checkpoint inhibitors (CPIs) of immunity, are used in the clinic for the treatment of various forms of cancer. Antibodies to CTLA-4 cause systemic toxicity and are approved only for some tumors. Antibodies against PD1 or PD-L1 have been successfully used for the treatment of various forms of cancer and are characterized by low toxicity. However, the response to therapy using CPIs is not always observed. The development of more effective approaches to cancer therapy based on PD1/PD-L1 inhibitors requires additional research. The aim of this work was to express the extracellular part of the murine PD-L1 protein (exPD-L1) and obtain antibodies to PD-L1. The mouse exPD-L1 protein was obtained and characterized in the bacterial expression system. exPD-L1 protein was used to immunize mice in order to produce anti-PD-L1 antibodies. Using hybridomic technology, 5 clones expressing antibodies to exPD-L1 were obtained. Antibodies of the B12 clone were developed in the ascitic fluid of BALB/c mice and purified by affinity chromatography. The ELISA method for purified antibodies showed specific binding to the exPD-L1 protein and the commercial protein of the extracellular part of murine PD-L1. Experiments using flow cytometry and confocal microscopy have shown that the antibodies obtained bind the intracellular form of the PD-L1 protein, unlike commercial antibodies binding the membrane form.

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M. S. Goryunova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

D. Y. Ryazantsev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. E. Petrova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

V. V. Kostenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Lomonosov Moscow State University

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Leninskie gory 1, Moscow, 119991

A. O. Makarova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Lomonosov Moscow State University

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; Leninskie gory 1, Moscow, 119991

R. V. Kholodenko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. V. Ryabukhina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

D. V. Kalinovsky

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

O. D. Kotsareva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. V. Svirshchevskaya

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Author for correspondence.
Email: esvir@mail.ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. Expression of exPD-L1 protein. (a) – Analysis of exPD-L1 gene expression in mouse cell lines; (b) – Schematic diagram of the pQE30 expression vector containing the exPD-L1 protein gene; (c) – Electropherogram of the PCR results for the gene fragment encoding exPD-L1 before cloning (1) and after insertion into the intermediate plasmid pTZ57R/T (2); (d) – Electropherogram of the results of exPD-L1 protein isolation and purification. (3) – E. coli lysate applied to a column with Ni-NTA agarose; (4) – Column breakthrough; (5) – Column wash fraction from non-specifically bound proteins; (6) – Elution fraction (in addition to the target protein, its degradation products are visible); (7) – Protein solution sample after reversed-phase chromatography. (8) – a sample of protein solution after protein refolding and dialysis; M – a marker of the molecular weight of proteins and DNA.

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3. Fig. 2. Characterization of monoclonal antibodies to exPD-L1 produced by the obtained hybridomas. (a) – Binding of exPD-L1 protein by antibodies from the culture fluid of the obtained hybridomas by ELISA; (b) – recognition of proteins of the extracellular part of PD-L1 (exPD-L1 and RPA788Mu01) and the comparison protein (phage Phi29 DNA polymerase with 6xHis tag) by mAbs isolated from the ascites fluid of B12 hybridoma; (c) – electropherograms of RPA788Mu01 protein and (d) purified B12 mAbs under reducing and non-reducing (from left to right) conditions; (d) – isotyping of mAbs secreted by the obtained hybridomas. (e) – recognition of B12 hybridoma mAb in dot blot of Phi29-6xHis phage DNA polymerase (1), exPD-L1 (2), and RPA788Mu01 (3) (decreasing protein concentration from left to right); (g) – electropherograms and Western blot of mouse B16/F10 (1, 3, 5) and human SCOV-3 (2, 4, 6) cell lysates labeled with B12 antibodies against mouse PD-L1 (3, 4) and against two forms of heat shock protein 70 (constitutive and inducible forms) (5, 6); (h) – histograms of distribution of living EL-4 cells stained with B12-Cy3 and (i) 10F.9G2-PE. Control histograms are marked in pink, cells after binding to antibodies are marked in blue; (k) – dot plots of living and (l) permiabilized B16/F10 cells, (g, abscissa) – labeled with B12 or 10F.9G2 (ordinate). Control histograms are marked in pink, cells after binding to antibodies are marked in blue.

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4. Fig. 3. Analysis of PD-L1 localization in B16/F10 cells cultured in 3D conditions. Cells were incubated on an anti-adhesive substrate for 20 h without (a, b) and with the addition of mouse IFN-γ (c, d), fixed, perforated and stained with B12 (a, c) or 10F.9G2 (b, d) antibodies. The insets show enlarged images of individual cells. The green arrow indicates the plasma membrane of cells, the blue arrow indicates the nuclear membrane. Cell nuclei are stained blue (b, d). Scale bar is 7–9 μm.

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5. Table 3. Homology of mouse (mecPD-L1) and human (hPD-L1) PD-L1 proteins*

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