(M0930-04-19) Genetically Engineered PD-L1 Targeted Exosomes for Immunotherapy of Doxorubicin Resistant Triple Negative Breast Cancers

Purpose: Immunotherapy has emerged as a promising avenue for cancer treatment, leveraging the body's immune system to target and eliminate cancer cells. Among various strategies, programmed cell death ligand 1 (PD-L1) targeting has shown remarkable potential in enhancing anti-tumor immune responses. The purpose of this study was to develop genetically engineered exosomes that can target PD-L1 in cancer as well as deliver payloads of anticancer agents.

Methods: Several surface markers that are differentially expressed in resistant TNBC cells such as MDA-MB-231 Doxorubicin (DOX) Resistant (RT), MDA-MB-231 Docetaxel (DTX) Resistant cell lines were screened with western blotting. For expression of anti-PD-L1 scFV, a sequence optimized peptide of 241 amino acids was engineered from available heavy and light chain sequences of Atezolizumab, humanized anti-PD-L1 monoclonal antibody. A recombinant pDisplay plasmid encoding tagged, anti-PD-L1 scFV, HA and Myc tag was constructed (ThermoFisher scientific) and was used to transfect HEK 293T cells to generate stable cell line. Selection of stable clones was done using geneticin over a period of 3 weeks. Expression of anti-PD-L1 was confirmed by western blotting through Myc tag probing since the anti-PD-L1 sequence used for vector construction was genetically fused with Myc sequence. Subsequently, these cells were transferred to PBS wheel bioreactor and exosomes were isolated using ultracentrifugation by our established procedures. Exosomes isolated were characterized for the expression of anti-PD-L1(PDL-E) through western blotting as well as Fluorescent Activated Cell Sorting (FACS). The PDL-E were also characterized by NTA, Fluorescent NTA as well as flow nanoanalyzer (NanoFCM Inc) for the number of copies of anti-PD-L1 expression relative to particles number of exosomes. PDL-E were screened for their ability to knockdown PD-L1 in breast (MDA-MB-231 DOX RT, MCJB70 PDX cell lines), lung (H1975 OSM RT), and pancreatic cancers (MIA PaCa-2). Following, these PDL-E were loaded with STAT 3 SiRNA by electroporation and the entrapment efficiency was determined by using Quickdrop (Molecular Devices). The STAT 3 SiRNA loaded exosomes (PDL-ESi) in combination with paclitaxel were evaluated in-vitro and in-vivo against TNBC MDA-MB-231 xenograft models. Western blotting and RT-PCR was done to understand the molecular mechanisms.

Results: Western blotting studies revealed the overexpression of surface markers such as PD-L1, EGFR, STAT 3, Nrf 2 in MDA-MB-231resistant cell lines relative to wild type cells. Following systematic transfection and selection studies, a stably transfected HEK 293T cell line expressing anti-PD-L1 was successfully generated and characterized with western blotting. Exosomes isolated were found to have a particle size of 112.5±3.5nm, particle concentration of 5.1E+10 Particles / mL and zeta potential of -32.22 ± 2.30 mV. Fluorescent NTA data showed 81.25% purity of exosomes. Western blot analysis demonstrated the expression of anti-PD-L1 on the surface of exosomes. It was further validated by FACS and flow Nanoanalyzer (NanoFCM Inc) which confirmed that 80% of PDL-E had expression of anti-PD-L1 after using various dilutions of the antibodies (Fig 1). PDL-E further demonstrated 40% (P < 0.001) of PD-L1 knockdown in both in-vitro and in-vivo breast cancer tumors and 70% (P < 0.0001) of PD-L1 knockdown in in-vivo H 1975 lung tumors. Also, PDL-ESi when combined with paclitaxel reduced the IC50 value of paclitaxel by 4 folds and 8 folds from 31.44nM±0.56 to 8.14nM±0.87 and 102.76nM±6.4 to 13.52nM±0.45 in 2D cultures and 3D spheroids respectively (Fig 2). In-vivo studies demonstrated that there was significant reduction in tumor volume by 3 folds of combination group (PDL-ESi and paclitaxel) in comparison to control and other groups (PDL-E, STAT 3, Paclitaxel, PDL-ESi). Western blot analysis demonstrated down regulation of PD-L1 (P < 0.001), PD-1(P < 0.001), mTOR (p < 0.0001), PARP (p < 0.001), STAT 3 (p < 0.0001), NFKB (P < 0.0001) and upregulation of caspase 9 (0.001). From the western blot analysis, it was evident that paclitaxel downregulated apoptotic markers such as mTOR, PARP significantly whereas PDL-ESi downregulated immune markers such as PD-L1, PD-1, NF-KB, STAT 3 (Fig 3). Hence this treatment approach includes both chemotherapy as well as immunotherapy for better efficacy.

Conclusion: PDL-E showed significant cytotoxicity and PD-L1 knockdown both in-vitro and in-vivo against doxorubicin resistant MDA-MB-23 cells. Further, PDL-ESi when combined with paclitaxel showed significant reduction in tumor volume. Western blot analysis further confirmed that these exosomes work by inhibiting PD-L1/NFKB pathway and finally lead to apoptosis in tumors. In conclusion, we developed exosomes that can act as drug delivery vehicles through active targeting as well as possess inherent immunomodulatory activity and can be used against a variety of tumors which overexpress PDL1(e.g. colon, lung, pancreatic, melanoma). These exosomes will be further investigated on humanized mice to optimize the benefits of this therapy. This is the first study to develop active targeted exosomes which possess innate anticancer activity through genetic engineering.


Figure 1 Generation of stable anti-PD-L1 cell line, isolation, and characterization of transfected exosomes. A) Western blot analysis of MDA-MB-231 wild type, MDA-MB-231 DOX RT, MDA-MB-231 DTX RT cell line. B) Represents construct of anti-PD-L1 plasmid C) Western blot represents expression of anti-PD-L1-Myc in HEK293T cells post transfection. C) Represents expression of anti-PD-L1 on the surface of exosomes isolated from transfected HEK 293T cells. D) FACS analysis showing anti-PD-L1 presence on the surface of transfected exosomes. E) Fluorescent NTA of transfected exosomes. F) NanoFCM analysis for quantifying the number of copies of anti-PD-L1 expressed relative to total number of exosomes.


Figure 2 In-vitro cytotoxicity assessment of transfected exosomes. A) Knockdown of PD-L1 in in-vitro MDA-MB-231 DOX RT cells after treatment with transfected exosomes. B) In-vitro 2D cytotoxicity assay of PDL-E, STAT 3, Paclitaxel, Doxorubicin, PDL-ESi and PDL-ESi + Paclitaxel in MDA-MB-231 DOX RT cell line. B) In-vitro 3D cytotoxicity assay of PDL-E, STAT 3, Paclitaxel, Doxorubicin, PDL-ESi and PDL-ESi + Paclitaxel in MDA-MB-231 DOX RT cell line.


Figure 3 In-vivo anticancer activity of transfected exosomes. A) Knockdown of PD-L1 in in-vivo MDA-MB-231 DOX RT breast tumors (P <0.001). B) Knockdown of PD-L1 in in-vivo H1975 OSM RT lung tumors (P <0.0001). C) Graph representing tumor volumes in MDA-MB-231 DOX RT Xenograft mouse model (P <0.0001). D) Western blot analysis of tumors isolated from mice.